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Synthesis and murine biodistribution of a para and ortho iodinated radiopharmaceutical directed towards… Alcorn, Laura N. 1997

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SYNTHESIS AND M U R I N E BIODISTRIBUTION OF A P A R A AND O R T H O IODINATED RADIOPHARMACEUTICAL DIRECTED TOWARDS T H E MUSCARINIC R E C E P T O R by L A U R A N . A L C O R N B.Sc. (Pharm.), The University of British Columbia A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Faculty of Pharmaceutical Sciences (Division of Pharmaceutical Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 1997 © Laura Nancy Alcorn, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada Date DE-6 (2/88) Laura N. Alcorn Muscarinic Radiopharmaceutical ABSTRACT Ortho and para N-(iodobenzyl)(cyclopentylmethyl)-t-butylamine (o-ICTB A and p-lCTBA) were synthesized and radioiodinated. The analogs were designed to bind specifically to the muscarinic receptor for use as radiopharmaceuticals in the diagnosis and monitoring of myocardial disease with standard nuclear medicine techniques. Uptake in receptor-bearing tissues was assessed by preliminary biodistribution studies in mice. The distribution of the muscarinic receptor is postulated to alter in response to disease. There may be an increase or decrease in the density and number of receptors. Receptors and their ligands typically interact with high sensitivity and specificity. The use of a radiolabeled compound which binds to the receptor could allow external imaging of its distribution by detection of the radioactivity in target tissue compared to non-target organs. This would permit early detection of changes in receptor density and monitoring of the onset and progress of disease. Cardiovascular disease remains a significant cause of morbidity and mortality in man. Techniques which assist in its early detection would be expected to significantly contribute to both prevention and treatment. The muscarinic receptor exists in at least five genomic forms. Various tissues typically express several muscarinic subtypes. This is not the case in the heart where predominantly only the ml subtype is expressed. Therefore, this m2 subtype if bound by a radiopharmaceutical, might be expected to demonstrate the health of the receptor population in the heart with little interference from background radioactivity in surrounding tissues. ii Laura N . A lco rn ^ L l L Muscarinic Radiopharmaceutical A simple, reliable synthetic method was devised involving five reaction steps. The overall yield for the synthesis of o - ICTBA was 39% and for p - I C T B A was 37%. Identity testing was done for o - ICTBA and p-ICTBA using normal and reverse phase thin layer chromatography, proton nuclear magnetic resonance spectroscopy and elemental analysis. Additionally, the ortho analog underwent Fourier transform infrared spectroscopy; positive electrospray ionization and atmospheric pressure chemical ionization mass spectrometry; high resolution mass spectrometry and; high performance liquid chromatography. Radioiodination using the Cu-I assisted exchange technique was used to prepare the final radiopharmaceuticals. The radiochemical purity for the ortho and para analogs was 98.8% and 98.9%), respectively. The radiochemical yields for each analog was 42% and 98.9%) for o - ICTBA and p-ICTBA, respectively. The specific activity of the final preparation was low at a maximum of 122 GBq/mmol (3.34 Ci/mmol). Biodistribution studies with each analog were done in mice. Ten tissues were assessed at 0.25, 1, 2, 4 and 6 hours post-injection. The following organs were harvested: blood, liver, kidneys, small intestine, adrenal gland, stomach, lung, heart, muscle and the tail. In an attempt to specifically block the muscarinic receptor, atropine was preinjected in a second series of mice who underwent an otherwise identical biodistribution protocol. Uptake in the selected tissues was compared with and without the receptor blocking ligand. iii Laura N . A lco rn Muscarinic Radiopharmaceutical The maximal heart to blood ratio obtained with the ortho derivative was 1.63 at 15 minutes post-injection and with the para was 1.54 at the same time point. These values are insufficient for efficient external imaging. For both analogs the highest target-to-nontarget ratio was observed in the adrenal gland at 4.35 for the ortho and 12.0 for the para. A marked difference in the distributions of the ortho and para analogs was observed. Organ uptake was consistently much higher with the para species. Atropine pre-injection did not result in notable attenuation of radioligand accumulation in receptor-bearing tissues after administration of the ortho analog. For the para species, uptake by the heart and brain was attenuated by approximately 40% at 0.25, 1, 4 and 6 hours post-injection. Laura N. Alcorn i—11 Muscarinic Radiopharmaceutical TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS... v LIST OF TABLES vii LIST OF FIGURES ix LIST OF SCHEMA... xi LIST OF ABBREVIATIONS xii ACKNOWLEDGMENTS xiii DEDICATION xiv Chapter 1. LITERATURE SURVEY 1 1.1 G-protein coupled receptors, characterization of the muscarinic receptor 1 1.1.1 Biochemical action 4 1.1.2 Distribution 10 1.1.3 Molecular Structure 14 1.1.4 Pharmacological Selectivity 19 1.2 The effect of pathology on receptor populations 20 1.3 Use of receptor ligands as radiopharmaceuticals for imaging 29 1.3.1 Stereochemistry ; 29 1.3.2 Nonspecific uptake 30 1.3.3 Effect of iodine position on the aromatic ring 31 1.3.4 Ligand affinity and receptor density 31 1.3.5 High specific activity radioiodination 32 1.3.6 Radioiodination methods 33 1.3.7 Muscarinic receptor radiopharmaceuticals 36 1.4 Chemistry of acylation and borane reduction reactions 52 1.4.1. Acylation reactions 52 1.4.2. Borane reduction 55 1.5 Statement of rationale and purpose 62 Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS 67 2.1 Preliminary research .67 2.2 Synthetic strategy .69 2.2.1 Reagents : 69 2.2.2. Equipment, instrumentation 70 2.2.3 Synthesis 71 2.2.4 Identity testing 74 2.3 Results and discussion 78 2.3.1 Synthesis : .78 2.3.2 Identity Testing and Radiochemical Analysis 81 Chapter 3. BIODISTRIBUTION IN MICE 94 3.1 Experimental 94 3.1.1. Animal protocols 94 3.1.2 Statistical Methods 95 3.2 Results and discussion 95 3.2.1 Unblocked Studies 96 3.2.2 Ortho versus Para Distribution 104 3.2.3 Effect of Atropine Blockade 105 Chapter4. SUMMARY. /. 109 Appendix 1. Spectral Analysis 112 Appendix 2. Summary of Biodistribution Results ....117 Appendix 3. Biodistribution Program 123 REFERENCES 128 vi Laura N . A l c o r n Muscarinic Radiopharmaceutical LIST OF TABLES Table 1. Localization of Peripheral Muscarinic Receptor Subtype mRNA and Receptor-Specific Immunoreactivity 11 Table 2. Distribution of Muscarinic Receptors in the Canine Heart 13 Table 3. Operational characterization of muscarinic receptor 20 Table 4. Summary of Investigations of Muscarinic Receptor Plasticity in Cardiac Disease Showing Decreased Density 28 Table 5. Summary of Investigations of Muscarinic Receptor Plasticity in Cardiac Disease Showing Increased Density 29 Table 6. Summary of Investigations of Muscarinic Receptor Plasticity in Cardiac Disease Showing No Change in Density 29 Table 7. Biodistribution of 123l-o-BIBTA at varying specific activities and with preinjection of atropine 68 Table 8. Analytical techniques Used in Identity Testing for ICTBA and precursors 82 Table 9. HPLC Retention Times for o-ICTBA, p-ICTBA and Free Iodine 86 Table 10. Physico-Chemical Properties of the Intermediates and Final Product for o-ICTBA 88 Table 11. Physico-Chemical Properties of the Intermediates and Final Product for p-ICTBA 89 Table 12. Atomic Composition Report for o-ICTBA following High Resolution Mass Spectrometry 91 Table 13. Atomic Composition Report for p-ICTBA following High Resolution Mass Spectrometry 91 Table 14. Biodistribution of QNB and ICTBA analogs 99 Table 15. Target-to-Nontarget Ratios with QNB and ICTBA analogs 100 Table 16. Adrenal:Blood Ratios with QNB and ICTBA analogs 102 vii Laura N . A lco rn Muscarinic Radiopharmaceutical Table 17. Murine Biodistribution of Ortho-123l CTBA (cyclopentyltertiarybutylbenzylamine) 117 Table 18. Murine Biodistribution of Ortho-123l CTBA (cyclopentyltertiarybutylbenzylamine) Post Atropine Blockade 118 Table 19. Murine Biodistribution of Para- 1 2 3l CTBA (cyclopentyltertiarybutylbenzylamine) 119 Table 20. Murine Biodistribution of Para- 1 2 3l CTBA (cyclopentyltertiarybutylbenzylamine) Post-Atropine Blockade 120 Table 21. Murine Biodistribution of Ortho-123l CTBA (cyclopentyltertiarybutylbenzylamine) 121 Table 22. Murine Biodistribution of Ortho-123l CTBA (cyclopentyltertiarybutylbenzylamine) Post Atropine Blockade. 121 Table 23. Murine Biodistribution of Para- 1 2 3l CTBA (cyclopentyltertiarybutylbenzylamine) 122 Table 24. Murine Biodistribution of Para- 1 2 3l CTBA (cyclopentyltertiarybutylbenzylamine) Post Atropine Blockade 122 viii Laura N . A l c o r n Muscarinic Radiopharmaceutical LIST OF FIGURES Figure 1. Acetylcholine 2 Figure 2. Mechanism of Activation and Action of G Proteins 4 Figure 3. "Baldwin model of the rat m3 muscarinic receptor 15 Figure 4. Muscarinic Receptor Structure and Functional Domains 18 Figure 5. IQNB 38 Figure 6. Bretylium 46 Figure 7. RIBA 46 Figure 8. lododexetimide 49 Figure 9. Benztropine 51 Figure 10. Diborane 58 Figure 11. Borane-THF 59 Figure 12. N-(iodobenzyl)(cyclopentylmethyl)-t-butylamine 65 Figure 13. o-BIBTA 67 Figure 14. Ortho- ICTBA 84 Figure 15. Para-ICTBA 85 Figure 16. o-ICTBA, Percent Injected Dose/gram Vs Time Post-injection (hr.): Heart Vs Blood .....97 Figure 17. p-ICTBA, Percent Injected Dose/gram Vs Time Post-injection (hr.): Heart Vs Blood 98 Figure 18. o-ICTBA, Percent Injected Dose/gram Vs Time Post-injection (hr.): Adrenal Vs Blood 103 Figure 19. P-ICTBA, Percent Injected Dose/gram Vs Time Post-injection (hr.): Adrenal Vs Blood 103 ix Laura N . A l c o r n .—11 Muscarinic Radiopharmaceutical Figure 20. P-ICTBA, Percent Injected Dose/gram vs Time Post-injection (hr.) in the Heart: Blocked vs Unblocked 106 Figure 21. P-ICTBA, Percent Injected Dose/gram vs Time Post-injection (hr.) in the Heart: Blocked vs Unblocked 107 Figure 22. O- ICTBA, Percent Injected Dose/gram Vs Time Post-injection (hr.) in the Lung: Blocked vs Unblocked 107 Figure 23. 200 MHz Proton magnetic resonance spectrum of ortho-ICTBA in CDCI3 112 Figure 24. 200 MHz Proton magnetic resonance spectrum of para-ICTBA in CDCI3 112 Figure 25. UV Recording of Reverse phase High Performance Liquid Chromatograph of ortho-ICTBA 113 Figure 26. Radioactivity Recording of Reverse phase High Performance Liquid Chromatograph of ortho-ICTBA 113 Figure 27. UV Recording of Reverse phase High Performance Liquid Chromatograph of para-ICTBA 114 Figure 28. Radioactivity Recording of Reverse phase High Performance Liquid Chromatograph of para-ICTBA 114 r Figure 29. Atmospheric Pressure Chemical Ionization Mass Spectrum of ortho-ICTBA 115 Figure 30. Positive electrospray Mass Spectrum of ortho-ICTBA 115 Figure 31. Fourier Transform Infrared Spectrum of ortho-ICTBA, neat ...116 Laura N. Alcorn Muscarinic Radiopharmaceutical LIST OF SCHEMA Scheme 1. Wallach Reaction 35 Scheme 2. Acylation Reaction 53 Scheme 3. Mechanism of Acylation Reactions 53 Scheme 4. Amine Acylation 53 Scheme 5. Formation of Acylammonium Salt 54 Scheme 6. Secondary amide reduction scheme 55 Scheme 7. Tertiary amide reduction scheme 55 Scheme 8. The reaction mechanism for hydride reduction of tertiary amides 56 Scheme 9. Mechanism for Diborane Reduction 59 Scheme 10. Formation of the Borane-Methyl Sulphide Adduct 60 Scheme 11. Reduction scheme for primary amides 60 Scheme 12. Reduction scheme for secondary amides. 61 Scheme 13. Reduction scheme for tertiary amides 61 Scheme 14. Synthesis of o-ICTBA 80 Laura N . A l c o r n LIST OF ABBREVIATIONS Muscarinic Radiopharmaceutical % ID/g percent injected dose per gram of tissue •AC adenylyl cyclase A C h acetylcholine BMS borane methyl sulfide i l - i3 intracellular loops 1 -3 in the muscarinic receptor IBTAmide iodobenzyl-terrbutylamide IBTAmine iodobenzyl-terr-butylamine kDa kilodalton M , - M 4 muscarinic receptor subtypes 1-4 characterized by pharmacological techniques m,-m5 muscarinic receptor subtypes 1-5 characterized by cloning techniques mAChR's muscarinic acetylcholine receptors ol - o3 extracellular loops 1 -3 in the muscarinic receptor o-BIBTA N-bis(o-iodobenzyl)-terr-butylamine I C T B A N-(iodobenzyl)(cyclopentylmethyl)-/-butylamine o-ICTBA N-(t9-iodobenzyl)(cyclopentylmethyl)-/-butylamine o-ICTBAmide N-( o-iodobenzyl)(cyclopentylmethyl)-t-butylamide P E T Positron emission tomography p-ICTBA N-(p-iodobenzyl)(cyclopentylmethyl)-/-butylamine p-ICTBAmide N-(/7-iodobenzyl)(cyclopentylmethyl)-t-butylamide P L C phospholipase C QNB quinuclidinyl benzilate RIBA c 9 r r / j ( 9 i o d o b e n z y l t r i m e t h y l a m m o n i u m iodide S P E C T Single photon emission computerized tomography T M transmembrane xii Laura N . A l c o r n Muscarinic Radiopharmaceutical ACKNOWLEDGMENTS The experience and knowledge I have gained in these past years in the masters program has been through the efforts and patience of several individuals. It has been a privilege to know my supervisor, Dr. Don Lyster for 18 years. From his example, I have learned what there is to know about the art of scientific investigation, experimental integrity, unshakable optimism in the face of repeated defeats in the lab and the joy of the learning experience. Some additional unexpected benefits have included the skills required to appreciate a truly fine chardonnay. Several others have contributed their time and knowledge and I would like to especially thank Dr. Hayes Dougan, Ms. Tami Lutz, Dr. Can V o , Dr. Mike Adam, Mr. Roland Burton, Mr. Simon Albon, Ms. Maggie Hampong, Dr. Rob Thies and the staff of the nuclear medicine department at Vancouver General Hospital. Additionally I am grateful for the efforts of my committee members: Dr. John Sinclair (chairman), Dr. Frank Abbott, Dr. Kath MacLeod, Dr. Alan Belzberg and Dr. Chris Mcintosh. I thank the University of British Columbia for the Graduate Fellowships I received. M y family as always, provided their love and support. Finally, thank you Dick for all that you do. xiii Laura N . Alco rn T J ^ T - Muscarinic Radiopharmaceutical DEDICATION This is dedicated to the memory of Dr. Barry Thomas Alcorn and everything you could have been, given half the chance. xiv Laura N . A l c o r n U Muscarinic Radiopharmaceutical Chapter L L I T E R A T U R E S U R V E Y Chapter 1. LITERATURE SURVEY Radiopharmaceuticals directed against the muscarinic receptor have been investigated only in the past twenty years1 , 2. To date, very few show promise for routine clinical use and of those which have been studied, uptake in target organs remains sub-optimal3. Much of the interest in receptor-binding radiopharmaceuticals is based on the premise that agents which are taken up in organ systems by virtue of function rather than architecture will provide much more valuable information about the health of the system under investigation. This is valid since in the early stages of disease, there is often little or no detectable change in the morphology or anatomy of the affected structures. However, at the point of detection, it is frequently difficult, if not impossible to successfully treat the offending pathology. Early detection therefore, has implications both economically as well as in maintaining the health of society. 1.1 G-protein coupled receptors, characterization of the muscarinic receptor A receptor may be defined as a structure, usually a protein, located on or in a cell, that responds to certain changes in the organism or its environment and transmits these responses to initiate a particular biological response or communicate a signal to downstream constituents in a biochemical pathway4. The signal is usually initiated by a molecule or ligand that binds with structural specificity to the receptor and may be referred to as the first messenger. Muscarinic receptors (mAChRs) comprise one of two classes of integral membrane cholinergic receptors (the other is the nicotinic receptor) 1 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y which act to mediate the effects of the parasympathetic nervous system5. The parasympathetic and adrenergic systems are the two major divisions of the autonomic nervous system which serve to regulate functions within the body that occur without voluntary control. The parasympathetic nervous system exerts excitatory and inhibitory control in both the central and peripheral tissues. The natural, or native first messenger for muscarinic and nicotinic receptors is the quaternary ammonium derivative, acetylcholine (ACh) (see Figure 1). C H 3 - C - O — ( C H 2 ) 2 - N + ( C H 3 ) 3 O Figure 1. Acetylcholine Stimulation of muscarinic receptors by A C h results in various peripheral physiological effects including decreased heart rate and force of contraction, contraction of smooth muscle, increased secretion from glandular cells and excitation or inhibition of neuronal activity6. Centrally, muscarinic actions include several vegetative, sensory and motor functions7. These are responsible for effects such as arousal, attention, rapid eye movement (REM) sleep, emotional responses, stress modulation and higher cognitive processes such as memory and learning. Thus, the action of a single neurotransmitter at specific receptors in the body results in the transfer of selective information to diverse systems. 2 Laura N . A l c o r n .—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y Complete characterization of this cholinergic receptor has begun only in the past 10 years. It is a glycoprotein of molecular weight 85,000 kDa and in the early 1980's, was purified to sufficient homogeneity from pig heart and brain to clone the m l and m2 subtypes8. Three additional subtypes have since been identified and cloned. Four of the five can be characterized using pharmacological techniques ( M l - M4). (See "1.1.4 Pharmacological Selectivity," page 19, for a discussion of pharmacological characterization techniques.) The five muscarinic receptor subtypes (ml to m5) allow for selectivity of acetylcholine's action. They possess common structural features which are shared amongst several receptor groups including rhodopsin, p and oc2-adrenergic receptors and substance K receptors. Additionally, these receptors belong to the superfamily of G protein-coupled receptors that are characterized by their interactions with specialized guanine nucleotide-binding proteins in the cell membrane prior to initiating their biochemical or electrophysiological effects. This process of binding to a receptor by an extracellular physical signal or first messenger and subsequent interaction with a G-protein to cause the intracellular response is called signal transduction. The muscarinic receptor may be characterized by its biochemical actions, distribution, molecular structure, and selectivity. These issues will be discussed in greater depth in the following sections with emphasis given to the cardio-specific m2 receptor subtype. 3 Laura N . A lco rn li Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y 1.1.1 Biochemical action. Figure 2. Mechanism of Activation and Action of G Proteins^ The use of G proteins as intermediaries in the pathway initiated by A C h and terminated by the particular biochemical event permits directionality, diversification and amplification of cellular communication. The G-protein complex is heterotrimeric, consisting of three nonidentical protein subunits (a, P and y) which are noncovalently associated and attached to the inner surface of the cell membrane (see Figure 2)9. Through a series of structural changes in their relative orientation, the complex acts on membrane-bound constituents which then initiate certain cellular pathways. The action of these constituents or effectors results in the biochemical effect. 4 Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y The signal transduction process involves the following steps: 1. In the "o f f or resting state (step A . , Figure 2) the three associated subunits of the G -protein are present in the cell membrane in the form of a complex. Guanosine diphosphate (GDP) is bound to the a subunit. 2. With docking of the extracellular first messenger at the receptor (step B. , Figure 2), a conformational change occurs in the receptor molecule which permits binding to the G-protein complex and subsequently, loss of G D P from the a subunit. 3. Once the a subunit is no longer associated with GDP, cytosolic guanosine triphosphate (GTP) readily occupies the empty binding site due to its relative greater intracellular abundance compared to GDP. This results in a conformational change in the a subunit, which activates it. 4. The activated G T P - a complex dissociates from the y and p subunits and diffuses within the inner cell membrane to link with the effector molecule forming the "on" state (step C . , Figure 2). The effector is often an enzyme such as adenylyl cyclase (AC) or phospholipase C (PLC) or it may be an ion channel. The effector molecule then may catalyze the production of further intracellular signaling molecules or second messengers such as cyclic A M P (cAMP). Alternatively, the activated a - G T P complex acts directly on an ion channel. There is also evidence that the P-y subunits may initiate some muscarinic effector functions including activation of P L C , A C and cardiac K + channels 9 1 0. 5 Laura N . Alcorn 7 ^ 1 L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y 5. The G T P moiety eventually undergoes hydrolysis to G D P via a-subunit associated-GTPase (step D., Figure 2). This results in inactivation of the a-nucleotide complex which turns off the biochemical pathway that was initiated. The energy released by G T P hydrolysis causes the conformational change in the a-subunit necessary for it to revert back to the inactive state. Thus, as with all G-proteins, the energy of hydrolysis is not involved with the downstream signaling process but is used to switch the signal off. Since the action of GTPase is effectively irreversible, this constrains the process to a single direction, unlike other forms of regulation such as allostery. Also, the rate of inactivation of the G-protein and thus duration of the biochemical effect is controlled by GTPase, rather than by dissociation of the first messenger from the receptor. It was originally thought that the rate of hydrolysis by GTPase determined when and for how long the transduction process was activated. However it has been found that the presence of the effector (e.g. Phospholipase C) may also increase the rate of hydrolysis of the complex from a half-time of about 1 minute by a factor of up to 50 times1 0. This is more compatible with the termination rate of muscarinic responses such as those on cardiac K + channels, at 135 min."1. 6. Once the a subunit is inactivated, it dissociates from the effector and reassociates with the free P and y subunits to reassume the resting state of the G-protein complex on the cell membrane. The p and y subunits appear to be structurally homogenous amongst different G proteins and different alpha subunits may be linked to an identical P-y pair or to different pairs. In contrast, the a chains confer uniqueness to the G-protein complex. The a 6 Laura N . Alco rn ,—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y subunit's identity determines which effectors and receptors the particular G-protein complex reacts with and has been described as the specific effector arm of the G-protein. It possesses the guanine nucleotide binding site as well as, in G-proteins linked to regulation of adenylyl cyclase, the site for bacterial toxin-catalyzed ADP-ribosylation. This latter effect is caused by the pertussis and cholera toxins and has been used as a major tool in identification of G-proteins. At least 21 different G protein a subunits are known to exist and have been characterized based on the biochemical process they are associated with9. A l l are products of separate genes. The divergent population of a subunits associated with G-proteins allows for diversity of signaling. The availability of multiple receptor types and first messengers also contribute to this phenomenon. Additionally, amplification of a first messenger's signal is possible due to G-proteins. This can be achieved through the ligand's ^ interaction with a single receptor which may then activate several different G-protein complexes or alternatively, one G-protein complex may activate several different effector systems. The specific biochemical effects of muscarinic receptors have been found to be subtype associated. The major pathways include the following three biochemical effects: 1. Inhibition of adenylyl cyclase (AC) activity. The main effect of muscarinic agonists in the heart is inhibition of A C and reduction of c A M P levels1 0. Adenylyl cyclase catalyses the formation of the second messenger, c A M P via elimination of pyrophosphate from A T P . Inhibition of the enzyme results in decreased intracellular concentrations of c A M P and decreased activity of c A M P 7 Laura N . A l c o r n U^A1L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y dependent protein kinase. Ultimately, phosphorylation reactions are reduced. It is thought that the positive inotropic and chronotropic effects of P-receptor stimulation are a result of cAMP-induced phosphorylation of regulatory proteins. The even-numbered muscarinic receptor subtypes, ml and m4 have been linked to this effect. The G, protein family is associated with this pathway and is pertussis toxin sensitive. 2. Stimulation of phospholipase C activity. Stimulation of this enzyme results in the production of inositol 1,4,5-trisphosphate (IP 3) and diacylglycerol (DAG) from hydrolysis of membrane-integral phosphatidylinositol bisphosphate8 1 0. D A G stimulates protein kinase C to perform phosphorylation of target proteins. IP3 acts by mobilizing calcium ions from intracellular stores. Various tissues possess receptors capable of this action, including the heart, gastro-intestinal smooth muscle and exocrine glands. The G-protein associated with this effector ( G q / G n ) is insensitive to the effects of pertussis toxin. The expressed products of the m l , m3 and m5 receptor genes appear to couple to P L C stimulation, while the m2 and m4 products couple much less or not at all. 3. Modulation of ion currents. Both potassium and calcium channels appear to respond to muscarinic stimulation". Inwardly rectifying cardiac K + channels, when activated by acetylcholine, cause hyperpolarization of sino-atrial pacemaker cells and a resulting slowing of diastolic depolarization. These channels appear to be distinct from the resting cardiac K + channels and both contribute to maintaining the resting negative membrane potential of the cell. With receipt of a depolarizing signal, they close and permit cell firing to occur. Pertussis 8 Laura N . A l c o r n ^ 1L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y sensitive G-proteins appear to be associated with transduction of this effect ( G / G 0 class). Also, no second messenger is invoked in this pathway. There is evidence that some muscarinic antagonists possess "negative intrinsic activity" on these channels and can reduce basal G-protein activity to cause their antagonist effect. This is in contrast to the usual mode of action of antagonists involving competition for the binding site on the receptor with the agonist. At calcium channels, muscarinic stimulation results in a rapid and transient spike in intracellular C a + 2 concentrations followed by a plateau phase. The transient phase is thought to be due to release of calcium from IP3 sensitive intracellular stores (see "2. Stimulation of phospholipase C activity" above). The plateau phase is provided by influx of extracellular calcium through a series of calcium channels. In excitable cells, decrease of the voltage-gated C a + 2 current leads to reduction in action potential duration and therefore, a negative inotropic effect. The effect is observed in atrial and ventricular tissue only when force has been increased by positive inotropes, although some evidence for inhibition of basal C a + 2 currents exists. Negative intrinsic activity by some muscarinic antagonists has been observed with this effect as well. Other voltage-insensitive channels found in non-excitable cells such as fibroblasts and epithelial cells are less well characterized. At least three groups have been described based on their association with second messengers and triggering signals'2. They are thought to be associated with the ml/m3/m5 grouping of muscarinic receptor subtypes. 9 Laura N . A l c o r n c=U L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y 1.1.2 Distr ibution. The muscarinic receptor is primarily found on autonomic effector cells innervated by postganglionic parasympathetic nerves13. These are found in secretory glands, smooth r muscle and the heart. They are also present in the brain, in ganglia and on cells which receive little or no cholinergic innervation. Thus, the receptor is distributed throughout most tissues in the body. The five subtypes are also heterogeneous in their distribution within a given tissue. A n exception to this is in the heart, where predominantly m2 receptors are found 1 0. The most reliable detection method for subtype distribution is by receptor specific antibodies10. Other approaches include detection of muscarinic receptor m R N A coding using radioactive hybridizing probes for the receptor specific nucleic acid. However, the presence of receptor-encoding genetic material doesn't always indicate that the receptor is expressed on the cell or, in the case of neurons, the location of the expressed protein may be very distant from the locale of the m R N A . Another method for determining subtype identity utilizes pharmacological profiles developed via radioligand-binding techniques (used to provide affinity measurements). Finally, functional studies may be done for subtype differentiation and agonist efficacy determinations. Table 1 summarizes the distribution of muscarinic receptor subtypes in the periphery as determined by antibody binding or m R N A detection. ; In the heart, the only muscarinic receptor found in appreciable amounts is the m2 subtype. Cardiac muscarinic receptors are associated with both cholinergic and adrenergic nerves. They are found on the efferent neurons of the cardiac ganglia where 10 Laura N . A lco rn r-^-11L Muscarinic Radiopharmaceutical Chapter ! . L I T E R A T U R E S U R V E Y they modulate ganglionic neurotransmission. These prejunctional receptors have inhibitory actions on neurotransmitter release. Postjunctional cholinergic receptors on the myocardium are responsible for the negative chronotropic and inotropic effects of muscarinic agonists. Table 2 gives the distribution of muscarinic receptors amongst the Table 1. Localization of Peripheral Muscarinic Receptor Subtype mRNA and Receptor-Specific Immunoreactivity t. Receptor m l m2 m3 m4 m R N A A b m R N A A b m R N A A b m R N A A b Exocrine glands X X X X X X Heart X X X X Vascular endothelial cells X X X Lung X X X X X X X X Ileum X X X X X X X X Uterus X Vas deferens X X X Sympathetic ganglion X X X X . . . . . . . . . . . x •• Adapted from ref. 10; X or X X refers to 10-49%, or 50-100% (respectively) immunoprecipitation o f 3 H - Q N B labeled solubilized receptors from indicated tissues. tissues of the canine heart. After labeling with the nonspecific antagonist Q N B , levels of mAChR's in rat heart are highest in the atria, sino-atrial node, atrioventricular node, bundle of His, and intrinsic cardiac ganglia. In situ hybridization histochemistry has also been done in rat heart using oligonucleotide probes labeled with 3 5 S for detection of m R N A 1 4 . Using this technique, less m2 m R N A than expected was found in the conducting system. Also, the intrinsic cardiac ganglia contained more than four times as much m R N A as the atria. The disparity could not be explained by expression of other receptor subtypes. The presence of a high density of prejunctional muscarinic receptors may have been responsible for the 11 Laura N . A lco rn T^LI L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y observations. The authors suggested that prejunctional mAChRs could have a more prominent role in regulation of cholinergic neurotransmission in the conducting system than was realized: In another study using quantitative light-microscopic autoradiography of radioligand binding sites, the spatial distribution of mAChRs in the sino-atrial node of canine hearts was determined15. The region of dominant pacemaker activity within the node was localized using in vitro mapping. Greater densities of both p,-adrenergic and muscarinic receptors were found in sino-atrial node tissues compared to adjacent right atrial muscle as well as in the dominant pacemaker regions compared to other sino-atrial regions. The muscarinic receptor population of the adrenal gland has not been as well characterized as that of the heart. The adrenal gland develops embryologically from two different tissues which give rise to the cortex (comprising 90% of its area in section) and the medulla. The cortex is made up of three strata: the zona glomerulosa, zona fasiculata and zona reticularis. The cells of the zona glomerulosa produce the mineralocorticoid hormones. Those of the zona fasiculata and reticularis produce glucocorticoid and sex hormones. The chromaffin cells of the medulla synthesize, store and secrete the catecholamines epinephrine and norepinephrine. The adrenal gland has been considered a homologue of the sympathetic ganglion1 6. Usually, preganglionic fibres of sympathetic ganglia release acetylcholine and the neurotransmitter of post-ganglionic fibres is norepinephrine. In the adrenal gland, the post-ganglionic fibres release epinephrine. The receptors in sympathetic and parasympathetic ganglia though, are primarily nicotinic. 12 Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y However, some muscarinic receptors have been found in the adrenal gland and stimulation of this organ as well as the usually is thought to be modulatory to nicotinic stimulation as well as mediating transmission by themselves. Table 2. Distribution of Muscarinic Receptors in the Canine Heart ^ Tissue Density (pmole/gram protein) Percent o f total cardiac receptors Left ventricular muscle. 37.1 28.9 Right ventricular muscle 52.7 15.2 Ventricular septum 58.1 21.7 Left atrial muscle 302 16.4 Right atrial muscle 200 17.8 . In human sympathetic ganglia, predominantly M l and less frequently, M2 m A C h R are present. Variability amongst species has been observed in studies of receptor subtype population in the adrenal gland. Bovine adrenal chromaffin cells have been reported to express M , or a heterogeneous mixture of M „ M 2 and M 3 1 7 . M 3 and M 4 have been reported in the rat1 8, M 2 in the cat1 9 and M , in the dog 2 0. In a recent study of catecholamine secretion by dispersed guinea pig chromaffin cells, M , and M 3 subtypes were implicated as being involved with epinephrine secretion and increased intracellular C a + 2 concentrations in the presence of extracellular calcium ions 1 7. These increases were attenuated by removal of extracellular calcium. In a study of the release of catecholamines evoked by splanchnic nerve stimulation in the dog adrenal gland, M , and M 2 receptor subtypes were involved with catecholamine release16. In the bovine adrenal medulla, 3 H - Q N B radioligand binding assays indicated that the M , subtype was the predominant muscarinic receptor21. 13 Laura N . A lco rn i—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y 1.1.3 Molecular Structure. (a) General Certain general principles apply to the structure of all integral membrane receptors. The segments of their three dimensional structure which span the cell membrane tend to exist in the form of a-helices of usually, 20-22 hydrophobic residues. There are usually hydrophobic interactions between the peptide sidechains and membrane lipids. The central hydrophilic cores of the helices take part in mutual hydrogen bonding. A l l members of the G-protein-coupled superfamily consist of seven a-helices arranged in a tight bundle within the cell membrane. (b) Muscarinic Receptor Recently, Baldwin et al described a model which maps the two-dimensional position of the seven helices of the m3 muscarinic receptor (Figure 3)2 2'2 3. The transmembrane domains (TMI-VII) are connected by three extracellular (ol-o3) and three intracellular loops (il-i3) (Figure 4). These loops and the cytoplasmic C-terminal domain are thought to be the result of strategically placed prolines in the helices. Amongst all the subtypes, the size of the loops is conserved at about 13-22 residues except for the i3 loop which is large, variable in size and least conserved. It is within the 7 transmembrane segments that maximal sequence homology amongst the subtypes is found. A comparison of the amino acid sequences of all the subtypes shows them to be approximately 40% identical overall, with 90% sequence identity within the transmembrane segments. Amongst 18 members of the G-protein superfamily, only 4% identity was found and only 19 amino acids were conserved6. Investigations of these 14 Laura N . A l c o r n ,—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y highly conserved residues through point mutagenesis has helped to gain insight into the function and structure of the receptors. J The receptor is a single chain peptide with an extracellular N-terminus of 21-65 amino acids and possessing 2-5 consensus sequences predictive of N-linked glycosylation. In other similar receptors, these glycosylated sites direct agonist binding. However in the m2 subtype, they have been found to be unimportant for ligand affinity or ability to take part in functional responses. The C-terminus possesses a highly conserved cysteine which, in other G-protein coupled receptors has been palmitoylated and may serve as a site of membrane attachment. This also has not been found in the muscarinic receptor. The intervening sequence between this cysteine and T M VII is well conserved 15 Laura N . A l c o r n .—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y and after the cysteine, the subtypes involved with phospholipase C stimulation (ml/m3/m5) have a long nonconserved sequence unlike the other subtypes. It is generally accepted that an aspartate residue in the 3rd transmembrane segment (TM) of all muscarinic subtypes (and also highly conserved among all biogenic amine receptors), is critical for high affinity binding 1 0. In a recent review, the results of several studies using mutagenesis to investigate the molecular structure of the acetylcholine binding site and the segments involved with G-protein coupling were summarized2 4. The receptor for acetylcholine as well as for other biogenic amines, is thought to possess a narrow cleft enclosed by a ring-like arrangement of the seven transmembrane helices, about 10-15 A away from the membrane surface. A n ionic bond is presumed to occur between the positively charged ammonium head group of the ligand and negatively charged T M III Asp 2 5 . Mutation of the Asp residue to Asn or Glu strongly reduces formation of the binary complex between acetylcholine and the receptor and its subsequent functional response. Although, ACh's binding is strongly inhibited, this is not as significant with other agonists. Ionic bonding may serve to anchor acetylcholine when bound to the receptor in its active conformation, but non-productive binding modes via non-polar forces may play a role as well, with other ligands2 6. Also, four Tyr and two Thr residues which are conserved among all muscarinic receptors and found in the "upper" portions of T M III, V , VI, and VII at about the same level as the aspartate residue are important for high affinity ligand binding. It is thought that they form a plane within which acetylcholine interacts. 16 Laura N . A l c o r n <—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y Hydropathy plots of the muscarinic receptor have indicated that they are analogous to the models of the opsins and adrenergic receptors. Three dimensional modeling of the muscarinic receptor has been based on the well-characterized bacterial membrane protein, bacteriorhodopsin. However, this model does not hold true in all investigations of the role of various conserved residues. Some discrepancies between experimental data and the predictions made by this model suggest that the three-dimensional structure of the muscarinic receptor differs from bacteriorhodopsin. O f particular interest has been the highly conserved T M VI Asn residue which according to the model should be intimately involved with acetylcholine binding but experimentally, its removal has no effect on agonist affinity or efficacy27. However, antagonist binding is significantly reduced. Structure-activity properties of ligands which bind to the receptor have also been investigated. The available antagonists have greater selectivity for specific subtypes than the agonists. Synthetic antimuscarinics were initially developed for therapeutic use from the prototype atropine28. However, there are other structurally unrelated compounds which demonstrate activity at the receptor as well. Most of the available compounds are either aminoalcohols, aminoalcohol esters or aminoamides. They generally possess a cationic site in the form of either a tertiary or quaternary nitrogen centre. Tertiary amines may gain access to the central nervous system, unlike the ionized quaternary species. Also, the quaternary compounds are thought to have greater activity at nicotinic receptors than the tertiary amine antimuscarinics29. It is believed that the cationic nitrogen moiety is involved with binding to the acidic (anionic) aspartate group located on T M 3 8. Some evidence that this anionic site is important for binding is from studies showing that 17 Laura N . Alco rn Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y protons behave as competitive antagonists and attenuate ligand binding to the receptor30-31. Intracellular glycosyla t ion selectivity of Himbacine and A Q - R A 741 selectivity o f U H - A H 37 selectivity of allostcric modulation by gal laminc G-prote in-coupl ing select ivi ty T T M I T M 3 T M 5 o3 select ivi ty o f agonists internal izat ion, sites o f phosphorylat ion by receptor kinase Intracellular Figure 4. Muscarinic Receptor Structure and Functional Domains^ 18 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y There is also evidence that binding may involve a H-bonding site on the ligand, 4.4 angstroms from the nitrogen head group3 2. This is not always present in ligands possessing high affinity for the receptor and may not be as critical as the aspartate binding site. Additionally, a methyl group located 5.7 angstroms from the nitrogen has been suggested to be essential for muscarinic agonist activity33. Agonist and antagonist structures differ most markedly in that bulky aryl or cyclic groups are usually present in 34 . . . the backbone of antagonists . Although both ligands usually contain either a tertiary or quaternary ammonium group, other structural features discussed above may not necessarily apply to both groups. 1.1.4 Pharmacological Selectivity. Five distinct muscarinic acetylcholine receptor subtypes have been identified by molecular biological techniques. The binding properties of the m l to m4 proteins after expression in eukaryotic cells, correspond to those of the pharmacological subtypes ( M l to M4) in the natural setting of brain and other tissues. Pharmacological characterization of the m5 subtype has not been achieved yet. Table 3 summarizes the current ligands available for characterization of muscarinic receptor subtypes using pharmacological techniques. Receptor subtype classification by pharmacological means relies on the development of selectivity profiles as given in Table 3 since there are no ligands which exclusively bind with high affinity to one subtype and not to any other. Thus, the binding behavior of a series of ligands must be measured in a particular tissue before the subtype distribution can be ascertained. The usual parameter that is measured is the antagonist 19 Laura N . A l c o r n • ,—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y equilibrium dissociation constant in pure and homogeneous receptor populations. These Table 3. Operational characterization of muscarinic receptors^S Antagonist Receptor subtype M , M 2 M 3 M 4 4 - D A M P 8.6(9.2) . 7.8 (8.4) 9.1(9.3) n.d. (8.9) Himbacine 7.2 (7.0) 8.5 (8.0) 7.6 (7.0) 8.8 (8.0) p-F . -HHSiD 7.2 (7.7) 6.0 (6.9) 7.9 (7.8) n.d.(7.5) Methoctramine 6.5 (7.3) 7.9 (7.9) 6.0 (6.7) 7.6 (7.5) Pirenzepine 8.3 (8.2) 6.8 (6.7) 6.9 (6.9) 7.7 (7.4) Tripitramine n.d. (8.8) 9.7 (9.6) 6.5 (7.4) n.d. (7.9)" The values are apparent affinities ( p K B values) determined functionally. Those values in parentheses are determined in radioligand binding studies at cloned human muscarinic receptors, expressed in C H O cells;-n.d. = not determined; 4-DAMP=4-diphenyl acetoxy N methyl piperidine methiodide; p -F -HHSiD=para fluoro hexahydrosiladifenidiol. are produced using transfected cell lines that express one particular receptor gene. However, these models assume that receptors in the cell line behave identically to those existing on cells in the intact organism. 1.2 The effect of pathology on receptor populations Alteration of receptor population or behavior in response to a pathological insult has been referred to as receptor plasticity. Reports of receptor plasticity may be found in association with disease36"40, in response to drug therapy 4 1 , 4 2, in alcoholism 4 3 , 4 4 and as part of the aging process45"47. Receptors play an important role in controlling human neuronal activity and the subsequent metabolic responses that result. The neurochemical mechanisms behind receptor plasticity and the resulting links with disease occurrence and progression remain incompletely understood. Evidence can be found in the literature for loss (down-regulation) 3 7 , 3 9 , 4 2, gain (up-regulation)40, a combination of loss and gain 4 8 as well as no change in receptor expression on the cell surface46. Additionally, investigators have demonstrated changes in expression of m R N A for specific receptor proteins49 and 20 Laura N . A l c o r n cJJ Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y modification of receptor coupling with the other components of signal transduction50'51. Treatment with the appropriate pharmacological agent has been shown in some studies, to reverse the abnormal receptor distribution 5 2 , 5 3. Some of the difficulties encountered in demonstrating a link between pathology and receptor population include extrapolation from animal models, standardizing the extent of disease, postmortem delays, use of radiolabeled native or synthetic agonists or antagonists, and diverse methods for quantifying receptor binding (both in vitro and in vivo). Receptors may be quantified in terms of their mass (absolute number per cell), or their density on the cell surface (fmol/mg or ml protein). They may also be assessed in terms of their ability to take part in the signal transduction process. It is conflicting whether plasticity is manifested by a change in receptor density, mass or function, or a combination of these parameters. There is evidence in certain disorders that receptor density decreases, but mass does not change38. Normal levels of the muscarinic receptor in the human heart have been estimated using positron emission tomography (PET) in combination with compartmental modeling. A P E T study in six normal human subjects used a noninvasive technique for quantifying muscarinic receptors in the heart54. A mathematical model and two-injection protocol with u C-labeled methylquinuclidinyl benzilate were used to calculate various kinetic parameters. The mean value of the receptor concentration, B ' m a x was 2 6 ± 7 pmole/ml tissue. Most of the work examining receptor plasticity in response to disease has been done in the brain, particularly in patients with Alzheimer's disease or animal models of Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y this disorder4 8*5 3'5 5'5 6 as well as in the aging process4 2'4 5'4 6. Other disorders including epilepsy3 9, diabetes37'50 and gastrointestinal diseases40'57 have also been associated with receptor changes. Multiorgan diseases such as diabetes may affect the receptor population in the heart although the underlying etiology of the disease lies within another organ. Depressed response by the diseased heart to anticholinergic agents such as atropine has been observed5 8. A study done in streptozotocin-diabetic rats showed the negative chronotropic effects of acetylcholine, carbamylcholine and bethanechol were amplified compared to normals and no change occurred in inotropic responses to these muscarinic agonists37. In the streptozotocin rats, the density of tritiated methylscopolamine binding sites was 34% lower in atria from the diabetic animals. The intent of this project was to develop a muscarinic receptor radiopharmaceutical, ideally specific to the m2 subtype. Since such an agent could find use for imaging the myocardium (a tissue containing predominantly this subtype), an in-depth discussion of the literature will be confined primarily to reports of receptor behavior in this tissue. Those studies finding decreased density in receptor population will be discussed first, followed by those reporting an increase and finally, no change in receptor distribution. Additionally, a paper examining the production of muscarinic receptor autoantibodies in Tetralogy of Fallot, is described. Table 4, Table 5, and Table 6 summarize these results as well as the study done in normal human heart tissue. Decreased Receptor Density. In a study of experimental cardiac hypertrophy in rats, two models were employed: the hearts of spontaneously hypertensive rats (SHR) and those of Wistar rats 22 Laura N . A lco rn .—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y with surgically induced abdominal aortal stenosis (ASR). Wistar Kyoto rats and sham operated Wistar rats were used as controls59. The density (fmol/mg protein) of muscarinic receptors in membrane homogenates of hearts from S H R was unchanged (127.6 +/- 11.5 vs 142.5 +/- 14.7), whereas in hearts from A S R it was found to be reduced (221.0 +/- 8.9 vs 308.8 +/- 16.1: all n = 6). The decreased receptor density was therefore only associated with massively hypertrophied hearts. Also, the compared groups were taken from two different strains which could compromise the validity of the conclusions. In a rat model of compensatory left ventricular hypertrophy, both the density (fmol/mg protein) and mass (fmol per left ventricle) of muscarinic and beta adrenergic receptors were measured38. Determinations were made 4-6 weeks after abdominal aortic banding. Competition curves were generated using a P-subtype-specific ligand and carbachol to determine the two subtypes of P receptors and the high and low affinity sites for both PJ and muscarinic receptors. The high affinity sites are thought to represent the receptors coupled to adenylyl cyclase. In left ventricular hypertrophy, the total density of muscarinic receptor sites decreased from 100±6 .8 to 7 8 ± 2 fmol/mg protein and that of high affinity receptors decreased from 6 3 ± 6 to 4 0 ± 4 . The total quantity of receptors per left ventricle remained unchanged, probably because the left ventricles were larger. The authors felt the decreased density was because either synthesis and degradation are augmented in parallel, or that receptor expression is not regulated during /eardiac hypertrophy and the gene encoded for the receptor is not activated. 23 Laura N . A l c o r n c=U L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y In a human study, the neuronal and amine receptor content of right ventricular myocardium in eighteen patients with Tetralogy of Fallot was investigated and compared to four acyanotic control patients with ventricular septal defect60. A l l patients had undergone an intracardiac repair within six months which included resection of the infundibular myocardium. Analysis was done on a surgical specimen obtained from the resected muscle of the parietal extension of the infundibular septum. Muscarinic receptors were quantified using 3H-quinuclidinylbenzylate as the radiolabeled antagonist in sarcolemmal membrane preparations. Nonspecific binding was subtracted. Receptor content was found to be increased in the control patients. The mean concentration was 968.7 pmole/mg protein ( S E ± 8 9 9 . 4 ) compared to 244.1 ( S E ± 1 1 8 . 8 ) in the Tetralogy of Fallot group (p = 0.002). This large variability in the data is a potential concern in interpreting the conclusions of the authors. In an animal study using tritiated quinuclidinyl benzilate, it was shown that heart failure was associated with a 36% decrease in muscarinic receptor density61. Mongrel dogs underwent aortal banding to induce heart failure. The density of receptors was decreased in the left ventricular sarcolemma of the heart failure dogs ( 3 . 6 ± 0 . 4 pmole/mg, n=8) compared to normal sarcolemma (5 .6±0 .6 pmole/mg, n=10). Agonist competition studies using carbachol and oxotremorine showed that it was loss of high affinity binding sites in the sarcolemma that accounted for the difference. The inhibitory pathway regulating left ventricular adenylate cyclase activity was also found to be defective. Addition of 1 p M methacholine inhibited adenylate cyclase stimulated activity by 15% in normal tissue but only 5% in tissues obtained from animals in heart failure, even after 24 Laura N . A lco rn urtLi 1 L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y correction for the already-existing reduced adenylate cyclase. Levels of the G T P -inhibitory protein known to couple the receptor to adenylate cyclase was measured with pertussis toxin labeling and were not found to be depressed in heart failure. In a rat study, heart rate variability (HRV) was investigated in rats with cardiac hypertrophy due to either cardiothyrotoxicity or aortic stenosis62. In normal animals, short and long oscillations in heart rate were sensitive, respectively to atropine and propranolol. They also correlated directly with heart rate. In the cardiothyrotoxic subjects, tachycardia was observed and, independently, an alteration in long oscillations. H R V was normal in compensatory cardiac hypertrophy due to aortic stenosis but the correlation between H R V and heart rate was not present. Since H R V is controlled by the sinus node and represents a balance between the sympathetic and parasympathetic nervous systems, the authors concluded that increased beta-adrenergic density and decreased muscarinic receptor density in thyrotoxicosis contributed to the observed decreased low frequency oscillations. Rat ventricle was used to determine if decreased receptor density in cardiac hypertrophy is transcriptionally or translationally regulated49. The absolute concentrations of mRNAs for both the receptor and the coupling proteins G(alpha s) and G(alpha 1-2) were quantified. Analysis of receptor density was done 5 weeks after aortal banding. A significant decrease in receptor m R N A levels in hypertrophied left ventricle, parallel to the reduction in receptor protein densities and negatively correlated to the left ventricular weight/body weight ratio was observed. The relative levels of G(alpha s) and Gfalpha 1-2) mRNAs remained unchanged and both accumulated proportionally to the 25 Laura N . A l c o r n i—' I Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y increase in left ventricular weight. This supports the hypothesis that receptor expression is pretranslationally regulated and the genes coding for G(alpha) and G (alpha 1-2) are activated during hypertrophy. Receptors and coupling proteins belong to two different groups of genes, which appear to be controlled by different regulatory processes. Increased Receptor Density. In a study of the effects of sepsis on muscarinic receptor distribution in rats, two subcellular fractions of rat myocardium underwent binding studies using [H-3]-quinuclidinyl benzilate ([H-3J-QNB) 6 3 . The sarcolemma (SL) and light vesicles (LV) , were compared. [H-3]-QNB binding studies showed that B m a x increased by 58.8% in SL with no changes in L V during early sepsis (9 hr. post), but there was no significant change in the K D value. The authors concluded that muscarinic cholinergic receptors in rat heart SL increase during early sepsis. No Change in Receptor Density. In a P E T study in dogs, chemical sympathectomy (via 6-hydroxydopamine) or surgical intrapericardial denervation was performed as a model for the denervation that occurs post cardiac transplantation64. Receptor levels were determined using a compartmental model. Control tissues contained 6 2 . 2 ± 1 0 . 4 pmole/ml tissue of muscarinic receptors, and this remained unchanged after chemical and surgical denervation. Beta-adrenergic receptor concentrations increased from 3 2 ± 4 pmole/ml to 6 2 ± 1 0 pmole/ml post chemical denervation and 6 9 ± 1 6 pmole/ml post surgery. After restoration of atrial innervation but not ventricular sympathetic innervation, muscarinic receptor density remained unchanged and beta receptor density remained high. 26 Laura N . A lco rn i—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y In another P E T study, the muscarinic receptor density in six human heart transplant patients was compared to that of six volunteers, five months after their surgery65. Evidence exists that decreased myocardial adenylate cyclase activity in response to guanine nucleotide stimulation occurs in denervated myocardium of transplant patients. Intravenous injection of "C-labeled methylquinuclidinyl benzilate ( M Q N B ) was given followed by generation of time-activity curves over regions of interest. A nonlinear mathematical model was used to calculate the concentration of muscarinic receptors. No difference was found between test and control subjects ( 2 4 ± 4 versus 2 6 ± 7 pmole/ml tissue, respectively). Other kinetic parameters including the association rate constant k+l5 the dissociation rate constant k, , and the equilibrium-dissociation rate constant were also unchanged. It was felt that the observed changes in signal-transduction function in transplant patients could not be linked to changes in receptor density and may be due to alterations in the G proteins coupled to adenylyl cyclase. The pathogenesis of idiopathic dilated cardiomyopathy is thought to involve cellular and humoral autoimmunity. In a human study, a peptide corresponding to the sequence 169-193 of the second extracellular loop of the human m2 muscarinic receptor subtype was used as an antigen to screen sera taken from patients with this disorder (n=36)66. They were compared to 40 healthy blood donors. O f the test patients, 38.8% were positive for antigen reactivity compared to 7.5% of normals. Affinity-purified autoantibodies from positive patients incubated with rat ventricular membranes resulted in a decrease in the maximal muscarinic receptor binding sites (B m a x ) but an increase in 27 Laura N . A l c o r n ,—' 1 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y K D in a concentration-dependent manner. Preincubation with atropine abolished the inhibitory effect of autoantibodies on receptor binding. Carbachol did not have this effect. Table 4, Table 5 and Table 6 summarize the findings of the aforementioned studies of muscarinic receptor behavior in myocardial diseases. Table 4. Summary of Investigations of Muscarinic Receptor Plasticity in Cardiac Disease Showing Decreased Density D I S E A S E T I S S U E S S T U D I E D O B S E R V E D R E S P O N S E T O D I S E A S E M E T H O D O L O G Y R E F normal human heart, ex vivo N / A ex v ivo , compartmental modeling 54 cardiac hypertrophy rat heart membrane homogenates decreased or unchanged muscarinic receptor density radioligand binding 59 compensatory left ventricular hypertrophy rat heart, ex-vivo decreased density but no change in absolute muscarin i d receptor numbers ex vivo, compartmental modeling 38 Tetralogy o f Fallot human infundibular septal tissue decreased muscarinic receptors radioligand binding 60 heart failure canine myocardial sarcolemma decreased muscarinic receptors radioligand binding 61 cardiac hypertrophy rat heart, ex vivo loss o f correlation between heart rate variability and heart rate, decreased low frequency variations ex-vivo heart rate monitoring 62 cardiac hypertrophy rat left ventricular tissue decreased muscarinic receptors, decreased receptor and coupling protein m R N A s slot blot analysis, binding studies 49 28 Laura N . A l c o r n il Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y Table 5. Summary of Investigations of Muscarinic Receptor Plasticity in Cardiac Disease Showing Increased Density D I S E A S E T I S S U E S S T U D I E D O B S E R V E D R E S P O N S E T O D I S E A S E M E T H O D O L O G Y R E F sepsis rat myocardium subcellular fractions increase in muscarinic receptors radioligand binding 63 Table 6. Summary of Investigations of Muscarinic Receptor Plasticity in Cardiac Disease Showing No Change in Density D I S E A S E T I S S U E S S T U D I E D O B S E R V E D R E S P O N S E T O D I S E A S E M E T H O D O L O G Y R E F . post cardiac transplantation (model) canine heart, ex vivo no change in muscarinic receptors ex-vivo compartmental modeling 64 post cardiac transplantation human heart, ex vivo ho change in muscarinic receptors ex v ivo, compartmental modeling 65 idiopathic dilated cardiomyopathy human, in vitro autoantibodies to the m 2 receptor in the blood in vitro, antibody screening 66 1.3 Use of receptor ligands as radiopharmaceuticals for imaging. 1.3.1 Stereochemistry. The design of a receptor imaging agent involves optimization of several physicochemical and biochemical properties. These include structural considerations which provide for adequate binding to the target receptor, choice of the radionuclide and labeling method. A radionuclide must be used which does not abolish the biological activity of the ligand. Labeling of receptor ligands for ex vivo S P E C T imaging has been done almost exclusively with radioiodine. A problem with the more commonly used 29 Laura N . A l c o r n .—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y "Technetium, is loss of receptor binding due to its large atomic radius and the effect of this on the ligand-receptor interaction. These interactions are typically stereoselective and use even of the smaller radioiodine is usually associated with appreciable loss of affinity by the ligand for the receptor. Stereochemical properties of the ligand are thought to be most significant in determining the dissociation kinetics of the interaction with the receptor30. This is based on the idea that specific, short range interactions between the ligand and receptor must be disrupted before dissociation occurs. Association kinetics are more influenced by physico-chemical properties such as hydrophobicity, size, and presence or absence of a N-methyl group. These factors influence the ligand's ability to approach and interact with the receptor. 1.3.2 Nonspecific uptake. A critical determining factor for the usefulness of any diagnostic radiopharmaceutical is achieving an adequate target-to-nontarget ratio. A ratio in the heart of at least 5 to 10 is desirable and greater than 20 is considered high 6 7 . This may be affected by serum half-life, blood pool and surrounding organ activity as well as blood supply and target organ binding. The ideal receptor-binding radiopharmaceutical should be taken up specifically and exclusively at the receptor of interest and not at other nonspecific sites. Additionally, it should not undergo loss of its radiolabel in the body throughout the time of imaging and its distribution should reflect that of the receptor population and not blood flow, nonspecific uptake, serum protein binding or other cell trapping processes. Cleavage of the radioisotope from the ligand is a particular problem with radioiodinated compounds (in vivo deiodination) . 30 Laura N . A l c o r n V - ^ I L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y 1.3.3 Effect of iodine position on the aromatic ring With the available labeling techniques, the position of the iodine moiety is usually limited to a site on an aromatic ring. Ortho -iodinated compounds may be more susceptible to the body's deiodinases due to their structural similarity to thyroid hormone6 8. As well, or//2o-labeled muscarinic binding agents have been associated with greater heart uptake compared to the brain 1' 6 9. Para labeled muscarinic radio-pharmaceuticals have been found to be more adrenospecific than ortho1. 1.3.4 Ligand affinity and receptor density. In order to achieve adequate target-to-nontarget ratios, the ligand must possess sufficient affinity for the receptor, (K a 109-101 0 M" 1) 7 0 . There must also be a high enough concentration of receptors in the tissue of interest. It has been shown in animals that the concentration of the muscarinic receptor in the base of the left ventricle is on the order of 5-20 pmol/g tissue, depending on the species71. The atria have the highest density of receptors but comprise only 10% of the weight of the myocardium7 0. The left ventricle should accumulate the largest portion of the total taken up by cardiac tissue due to its larger mass. In humans, the muscarinic receptor density is about 22.5 pmol/ml tissue or 275 pmol/g protein65. In an average human heart of 400 ml total tissue, this would result in 9,000 pmol of receptors. This represents a great excess of receptors compared to the number of radioligand molecules that would be available to the target organ. In this project, each mouse received approximately 900 pmol of ligand The development of these agents has been hampered by difficulties with interspecies variation in biodistribution patterns and the availability of sufficiently 31 Laura N . A l c o r n ^ I L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y subtype selective ligands3. Finally, the time to imaging should be within the constraints of a clinical setting and accommodate the decay characteristics of the radiolabel. This requires a rapid decline in radioactivity associated with background structures such as the surrounding organs and blood. Concomitantly, persistence of radioactive uptake at the target organ must occur. 1.3.5 High specific activity radioiodination. The specific activity of a compound may be defined as the radioactivity per mole or unit weight of radionuclide or labeled compound 7 2. In the case of radionuclides, the maximum theoretical attainable is determined by the decay constant. There is an inverse relationship between the magnitude of the specific activity and half life. In the case of 1 2 3I, the half life is 13.1 hours and the maximal specific activity is 239,876 Ci/mmol. For radiolabeled compounds, the theoretical maximum will be achieved if every potential labeling site on the molecule is labeled with the radionuclide of interest. This will be determined by the specific activity of the stock radioisotope solution and the efficiency of the labeling method. A low specific activity radioisotope solution will result in the addition of both l 2 7 I and radioiodine to the molecule of interest. Labeling methods may be either carrier or "no-carrier-added". Carrier is the term used to refer to stable ( , 2 7I), present or added in the radiolabeling procedure. In the case of "carrier-added" methods, 1 2 7I may be present either in the starting compound or is added as a contaminant in the radioisotope solution. If it is present in the starting compound, it is impossible to separate labeled product from non-radioactive precursor using physico-chemical means. In "no-carrier-added" methods, stable iodine is only present in tiny / 32 Laura N . A l c o r n ^ I L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y amounts due to its natural ubiquity in the environment and the starting compound has some other atom at the site destined for radiolabeling. This allows for easy separation of labeled compound from unlabeled precursor, usually by H P L C means. Thus, it is the purification step which provides high specific activity to the final product, rather than the labeling procedure. It is important that a receptor binding radiopharmaceutical have high enough specific activity to allow efficient detection of its distribution using external imaging techniques. This is determined by biochemical and physiological considerations such as the number of moles of receptor in the target tissue and the binding properties of the ligand. Generally, the number of target binding sites available to it following intravenous injection are relatively small 2' 7 3 , 7 4 . As the population of unlabeled ligand increases, this species competes with the labeled species for the available binding sites and the resulting image may have a poor or unacceptable target-to-nontarget ratio. 1.3.6 Radioiodination methods. Radioiodination methods are usually either electrophilic or nucleophilic. Often, the reaction mechanism cannot be proven in the laboratory due to the micromolar amounts of reagents involved. Rather, it is inferred empirically from what is known about the behavior of gram quantities of the reactive species. Electrophilic methods may be via aromatic or organometallic substitution. Classically, this approach has been used for the preparation of receptor radiopharmaceuticals. The proposed mechanism involves oxidation of the iodide ion (I) to the positively charged iodous ion which exists in the form of hypoiodite (HOI +). This 33 Laura N . A lco rn ! L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y ion acts as the reactive species in an electrophilic substitution reaction with the compound of interest. In proteins, this is typically the phenolic ring of a tyrosine amino acid or the imidizole ring of histidine. In other non-protein compounds, aromatic hydrogens are usually used in the substitution reaction. Several oxidizing systems are available and the choice is usually made based on preservation of biological activity in the ligand after exposure to sometimes harsh oxidizing conditions. Iododestannylation is an organometallic substitution method which has been used to produce labeled compounds with sufficient specific activity for receptor imaging. One approach for preparation of the alkylstannylated precursor is through reaction between an organic halide and hexa alkyldistannane in the presence of a palladium complex. Oxidative addition of the organic halide and bis trialkyl stannane occurs to the palladium complex (II). This is followed by reductive elimination of one of the trialkyl stannanes and the other is bound to the organic compound from the resulting Pd IV complex. The advantage of this approach is that the reaction mixture prior to addition of the radioiodine contains no ligand labeled to stable iodine. Iodination proceeds via electrophilic attack by iodine in the oxidized state on the aryl-tin bond. This occurs selectively in the presence of alkyl-tin bonds. Oxidants that have been used include chloramine-T and N -chloro-succinimide7 5. The final products are radioiodinated ligand and remaining stannylated ligand. This results in a simple purification step to remove unlabeled precursor. For biological tracers however, these methods do not always provide high enough specific activity7 1. 34 Laura N . A l c o r n L ^ U L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y The Wallach triazene approach is "no-carrier-added" and has been used fairly extensively for radioiodination of Q N B and its analogs76. In this approach (see Scheme 1) the position on the aromatic ring which is intended for attachment of the radioiodine is prepared as the amino derivative. The diazonium salt is then produced and coupled with 3-methylpiperidine to form the triazene derivative. Incubation of this species with high specific activity radioiodine at p H 5.5 and temperature 78°C for 50 minutes has resulted in a radioiodinated Q N B analog with 7.5% radiochemical yield 7 6. This low yield is the major drawback with the triazene approach. Scheme 1. Wallach Reaction A r N H 2 • A r N + 2 ^ - 2 A r — N = N - N R 2 - ^ - » - A r X N a 2 C 0 3 X" X = F, CI, Br, I Nucleophilic exchange using iodide ion (I) has also been used to provide a product with sufficient specific activity. A n example of this approach is the Cu(I) catalyzed method 7 7. Higher specific activity than with the triazene method (used for Q N B ) has been reported78. In this reaction, nucleophilic attack by iodide ion occurs. Tin (II) is added to maintain the iodine in its reduced form and prevent the formation of iodates and periodates during the heating process. The precise mechanism has not been completely characterized but is thought to involve formation of the cuprous ion (Cu + I ) , which is the soluble active form. Subsequently, an in-situ copper-organo complex forms with the ligand (labeled with stable iodine). Thus, the reaction is not "no-carrier-added". Ascorbic acid is used to cause formation of cuprous ion (Cu + 1) from cupric (Cu + 2 ) 7 9 . The advantage of ascorbic acid over other cuprous salts such as the bromide or chloride is that 35 Laura N . A l c o r n r^ -11L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y the catalytic species is present in solution. Insoluble radioactive cuprous iodide may be formed in the reaction, particularly in the presence of oxygen or hydroxide ions. A "no-carrier-added" approach is use of the bromo or chloro precursor for interaction with the cuprous ion in the presence of radioiodide ion. A method such as H P L C may then be used to separate radiolabeled from unradiolabeled product. In summary, the goal is to synthesize a compound with high affinity, specificity and selectivity as well as Jow non-specific binding. 1.3.7 Muscarinic receptor radiopharmaceuticals. The following sections summarize the pre-clinical and clinical experience with muscarinic imaging radiopharmaceuticals. Very few potential muscarinic receptor imaging agents have been identified to date. Common approaches used to investigate these agents include the following 1. Comparison of abnormal to normal tissue within the same subject or between diseased and healthy individuals. 2. Use of blocking ligands (antagonists) to block uptake of the test ligand at the receptor site. 3. Comparison of the radiolabeled receptor ligand to a non-specific radiopharmaceutical which shows blood flow or to the stereoisomer of the test compound which should distribute to non-specific binding sites only. Parameters which have been found to affect target organ localization include the stereochemistry, membrane permeability and lipophilicity of the ligand as well as choice 36 Laura N . Alcorn V - ^ IL Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y of isotope, labeling technique and position of the radiolabel. Variability in target organ uptake amongst species has also been a problem in identifying potential receptor-binding candidates. Four major groups which have been investigated include quinuclidinyl benzilate (QNB) and its methylated and propenyl analogs, benztropine, bretylium and dexetimide. O f these, Q N B and its analogs have probably been investigated the most extensively and found limited clinical application in human imaging of the brain 1 2 , 5 4 ' 5 6 , 6 5 . Low heart to lung ratios have limited QNB's usefulness as a myocardial imaging agent. This has been somewhat overcome by the use of the methylated analog, M Q N B . Benztropine has been studied as a potential P E T agent for the brain and although promising in the baboon, studies in humans have revealed this agent to be very slowly cleared from background structures, delaying the time to imaging and limiting its usefulness with the short lived positron-emitters. Bretylium analogs showed a high degree of concentration in the myocardium of rats, dogs and pigs. However, neither humans nor monkeys showed myocardial accumulation and further investigation since the early eighties has not been done. 1.3.7.1 Quinuclidinyl benzilate (QNB) and derivatives. Q N B (1-azabicyclo [2.2.2]oct-3-yl a-hydroxy-a-phenyl-oc-phenylacetate) is one of the most potent but non-specific muscarinic antagonists. Recently, there has been some autoradiographic evidence for possible m2 selectivity80. As well, biodistribution patterns suggestive of the m2 subtype have been demonstrated by ex vivo imaging of the radioiodinated derivative. The radiopharmaceutical has been used most successfully for assessing the distribution of muscarinic receptors in the brain (see Figure 5 for the 37 Laura N . A l c o r n ,—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y structure of IQNB). Persisting high levels of background radioactivity have limited its usefulness for imaging the heart. Unlike other muscarinic radiopharmaceuticals, the Q N B analogs have not been associated with decreased target-to-nontarget ratios in humans as compared to animals. Poor radiochemical yield and long delays to imaging due to slow clearance of background radioactivity are some problems that have been encountered with this group. Normal distribution of the radiolabeled ligand in the brain includes the basal ganglia, occipital cortex and insular cortex. Little uptake is seen in the thalamus and none in the cerebellum8 1. This corresponds to the relative distribution of muscarinic receptors in the normal human brain. Figure 5. IQNB Two other analogues besides the parent compound have been studied including IQNP (1-azabicyclo [2.2.2]oct-3-yl a-hydroxy-a-(l-iodo-l-propen-3-yl)-a-phenylacetate) and M Q N B (methyl quinuclidinyl benzilate). Replacement of Q N B ' s phenyl ring with an iodopropenyl substituent results in IQNP. M Q N B is the methylated 38 Laura N . A l c o r n ur=^ IL Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y quaternary derivative of Q N B . It is also known as clinidinium bromide and is used therapeutically as an antispasmodic in the drug Librax®. The work with radioiodinated Q N B derivatives has revealed several factors which determine optimal target organ localization. These are presented in the following brief descriptions. Effect of Radioiodination. Flanagan et al in 1979 investigated the kinetics of Q N B in rat synaptic plasma membrane-enriched fractions2. Three ligands were compared including Q N B , a para hydroxyl derivative and the radioiodinated para hydroxyl analogue. Iodination of the para hydroxyl analogue was done at the meta position of one of QNB's phenyl rings to form 3-quinuclidinyl-(3-iodo-4-hydroxy-benzilate (I-OH-QNB). This was compared to the uniodinatedpara-hydroxyl-QNB (OH-QNB) and the parent compound, Q N B . Displacement experiments using [ 1 2 5 I]OH-QNB gave the following I C 5 0 values: 1.5 x 10 -9 -10 M , 4 x 10 M and 5 x 10 M for I -OH-QNB, O H - Q N B and Q N B respectively. Thus, only a slight reduction in affinity for the muscarinic receptor was observed with I -OH-Q N B compared to O H - Q N B . Parallel binding studies in rat brain subcellular fractions gave similar results with the tritiated and iodinated species. Also comparable, were the concentrations of muscarinic antagonists required to block binding of either labeled agent. High specific radioactivity was obtained by exchange labeling. Stereochemistry. The stereochemistry of Q N B was found to be important for receptor binding in the work of Gibson, Reba and Rzeszotarski 7 6' 8 2 , 8 3. They began investigations of Q N B as a 39 Laura N . A l c o r n V-^11L Muscarinic Radiopharmaceutical Chapter V. L I T E R A T U R E S U R V E Y potential radioimaging agent in the early eighties. The parent structure exhibits stereoisomerism about the 3-carbon atom in the quinuclidine portion of the molecule. Iodination results in a second chiral centre. The receptor binding and target tissue uptake properties of the stereoisomers were determined in rat heart. R,R-4-[ 1 2 5I]IQNB was taken up by the heart more avidly than R,S-4-[ , 2 5I]IQNB (0.32 and 0.15 % ID/g respectively, 2 hours post injection)84. Also, the tritiated analogue (R-[ 3 H]QNB) was much more localized in the heart than either iodinated compound (3.56 % ID/g, 1.5 hours post injection)85. Addition of the hydrogen isotope has no effect on the stereochemistry of the ligand compared to addition of radioiodine. The binding kinetics of (R)-[ 3H]QNB and R,R and R,S-4-[' 2 5I]IQNB stereoisomers were also determined in transfected cell membranes expressing one muscarinic receptor subtype86. With (R)-[ 3H]QNB and the R,R iodinated stereoisomers, the on-off rate at the m2 subtype was greater than for the m l and m3 subtypes. Isotope Position and Lipophilicity The importance of isotope position and its effect on lipophilicity was demonstrated by Gibson et al. who measured the binding affinities of various halogenated Q N B derivatives in rat heart and brain 8 7. The meta or para fluorinated analogues did not display altered binding kinetics, but bromination or iodination of Q N B reduced affinity for the receptor in rat ventricle. Lipophilicity has been inferred from the transport rate constant across membranes and this was compared in rat brain capillaries amongst radioiodinated Q N B , tritiated cyclofoxy (an opiate receptor antagonist) and the blood flow indicator, 1 4 C iodoantipyrine88. The mean influx extraction ratio was highest for 40 Laura N . A l c o r n .—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y iodoantipyrine and lowest for IQNB. The authors felt that transport was highest for the least lipophilic analogue (iodoantipyrine) and decreased with increasing lipophilicity (cyclofoxy followed by IQNB). Binding of the lipid-soluble radioligands to plasma proteins and red blood cells was thought to prevent transport across the blood-brain barrier. This is in conflict with another commonly held view that lipophilicity favors transport across brain capillaries and is directly proportional to movement across the blood-brain barrier72. In an attempt to reduce QNB's lipophilicity and improve transport, one of the phenyl rings was replaced with either a cyclohexyl, a cyclopentyl or an n-butyl moiety8 9. Affinity for ventricular muscarinic receptors was not significantly affected by these modifications and the cyclopentyl analogue displayed the highest affinity for the receptor. Replacement with an alkoxylalkyl moiety however, reduced receptor affinity with the oxygen in the beta-position. Also, bromination of the second phenyl ring greatly reduced affinity, unlike bromination of the parent compound, Q N B . Addition of an ether linkage between the two phenyl groups resulted in the rigid structure, 3-quinuclidinylxanthene-9-carboxylate(QNX), which displayed considerably less affinity for ventricular muscarinic receptors90. However, in the caudate/putamen, binding was unaffected. Specific activity Specific activity is a particular concern with receptor imaging. The population of receptors available for binding by the radiopharmaceutical in the intact organism is much smaller than is the case with in vitro use9 1. It becomes critical that all available sites be bound by ligand bearing the radioactive tracer. With decreased specific activity, the 41 Laura N . A l c o r n .—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y number of unlabeled ligand molecules is increased as well as the number of receptor sites occupied by nonradioactive ligand. The end result is a much poorer target-to-nontarget ratio6 7. A less significant concern with low specific activity preparations is the risk of pharmacological effects with very toxic receptor binding compounds. The problem of specific activity with IQNB was addressed by Owens et al in 199278. The copper (I) assisted nucleophilic exchange technique was used to prepare a high specific activity product without the purification steps required with the triazene approach. The authors were able to show unique distribution of the radiopharmaceutical in patients with Alzheimer's disease as compared to controls. Also, the distribution of the receptor imaging agent was distinct from that of a blood flow indicator, 9 9 m T c H M P A O . The latter agent is expected to show smaller deficits in regions of disease than are demonstrated by the receptor agent. This is based on the idea that regions of early disease may have a normal blood supply despite early abnormal changes in receptor density. These regions would likely be found on the periphery of disease centers and this would give the larger deficits of the receptor agent's scan compared to that of the blood flow agent. Patients were given 185 M B q o f 1 2 3 IQNB in 5ml 20% aqueous ethanol and S P E C T imaging was done 21 hours post injection. In 1993 Owen's group did a more extensive clinical trial comparing the distribution of 1 2 3 I Q N B to that of 9 9 m T c H M P A O in eight patients with Alzheimer's dementia92. The Alzheimer's patients were compared to four elderly volunteers. The same Cu(I) assisted nucleophilic aromatic exchange reaction was used for radiolabeling. Each patient received 160 M B q of the radioligand (specific activity 14,800 GBq/mmol: 42 Laura N . A lco rn V - ^ IL Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y dose of ligand 90 ng/kg). S P E C T imaging was done at 21 hours post-injection. Six patients exhibited abnormal IQNB uptake which was no more remarkable than the defects displayed by the perfusion agent. Only two severely affected patients demonstrated deficits that were more extensive. The authors concluded that detectable reductions in receptor density following IQNB imaging occur only in end-stage disease. However, in a comparison of IQNB to P E T imaging with fluorodeoxyglucose (for detection of regional cerebral blood flow), defects in tracer uptake by the receptor imaging agent were larger than those demonstrated by F D G 8 1 . These differences could be explained by decreased muscarinic receptor binding or reductions in regional cerebral blood flow or cerebral atrophy. Comparison between S P E C T and P E T imaging must be done cautiously however, due to slight differences in tomographic resolution and possible scanning at different times in the course of the disease or in different patients. However in comparison to 9 9 m T c - H M P A O which presumably causes the same partial volume sensitivity, larger defects are still seen. In another clinical study, Weinberger et al. used S P E C T to follow the distribution o f 1 2 3 IQNB in fourteen patients with dementia and eleven healthy controls56. Imaging was only possible at 21 hours post injection of 185 M B q due to the need for background blood clearance of tracer. This long delay to imaging is frequently encountered with radioiodinated Q N B analogs in humans and can result in the need for larger doses of the short-lived isotopes. Eight of the 12 patients with Alzheimer's disease had focal defects in either the frontal or posterior temporal cortex. Two patients with Picks disease had frontal and anterior temporal defects. Four patients with Alzheimer's disease showed no 43 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 17 L I T E R A T U R E S U R V E Y focal defects. A concern with interpretation of the results was the uncertain contribution by brain atrophy and cell loss to the observed focal reductions. Methylated QNB The methylated analog of Q N B is a quaternary compound (unlike Q N B ) and is therefore membrane impermeant, binds only to cell surface receptors and is not metabolized. Therefore, improved target-to-nontarget ratios in the periphery should be possible. In 1979, Gibson et al compared the distribution of tritiated Q N B and its methiodide salt ( M Q N B ) in the rat, guinea pig and rabbit70. Heart uptake was 0.5 to 2.0 % ID/g with both radiopharmaceuticals. Heart-to-blood and heart-to-lung ratios were approximately 30 and 4, respectively. Preinjection with atropine, a muscarinic receptor blocking agent, attenuated tissue uptake by 89%. M Q N B gave superior results for the time to peak heart uptake and heart-to-blood ratio in the guinea pig and rat compared to Q N B . In two rabbits, uptake of M Q N B was found to be greatest in the atria. However, the total number of receptors in the left ventricle is 29 % and 22 % in the ventricular septum due to the relatively greater mass of these tissues. The binding kinetics of the R- and S-enantiomers of Q N B and M Q N B were compared in vitro amongst the m l , m2 and m3 receptor subtypes'8. Both the dissociation and association rate constants were determined. Variability amongst receptor subtypes was found in the dissociation rate constants of (R)-QNB and (R)-methyl-QNB but the association constants did not differ significantly. Delforge et al. have done considerable research with methylated Q N B and P E T imaging to noninvasively quantitate myocardial muscarinic receptors54'64'65. Two reports 44 Laura N . A lco rn i—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y have been summarized in a previous section 5 4 , 6 4 (see page 20, section 1.3 The Effect of Pathology on Receptor Populations). In normal humans, using a region of interest over the left ventricle, the mean receptor concentration B m a x , was 26 ± 7 pmole/ml tissue. This is comparable to the result obtained with 3 H - Q N B in humans93. The concentration was 275 pmole/g protein which is equivalent to 22.5 pmole/ml tissue based on a protein-to-tissue ratio of 12.2. The iodo-propenyl derivative of Q N B , (QNP) was synthesized by McPherson et al. in order to develop a muscarinic ligand with high affinity like Q N B , but improved labeling efficiency and decreased time to imaging, post-injection94"96. Q N B is labeled using the triazine decomposition method with only 20% radiolabeling efficiency and is cleared slowly from the body due to nonspecific binding. Replacement of one of the phenyl rings with the propenyl moiety permits labeling using the iododestannylation approach. This method has resulted in a higher radiolabeling efficiency (> 60%) and specific activity (55.5 GBq/umol) due to the production of almost no ligand labeled with nonradioactive iodine. IQNP contains two chiral centres like 1 2 3 IQNB, and can exist in the cis or trans orientation, due to the iodide on the double bond of the propenyl group. McPherson has found that the (E)-(-)-(-)- and (Z)-(-)-(-)-IQNP isomers have the highest affinity for membranes containing the m l , m2, and m4 subtypes ( K D i n the m l subtype, 0.383 and 0.13 n M , for the (E)-(-)-(-)- and (Z)-(-)-(-) isomers, respectively). In rat tissues, preblocking with Q N B prevented ligand uptake. The (Z)-(-)-(-) isomer showed promise as an m2 receptor imaging agent with a heart to blood ratio of 8:1, heart to lung ratio, 2:1 and heart to liver ratio also 2:1, four hours post-injection. Metabolic 45 ( Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y investigations in rats suggested that only unmetabolized E-(R,R)-IQNP is found in the brain up to 24 hours after intravenous administration. Organic extracts from the heart contained approximately 85% of unmetabolized IQNB as shown by T L C and H P L C analyses. Additionally, two minor radioactive components were also detected, one more polar and one less polar than IQNB. These slowly increased over time and were thought to represent metabolites of IQNB although their identity was not determined. Metabolic products and not the parent compound, were found in the liver, urine and feces. 1.3.7.2. Bretylium derivatives. Bretylium ( Figure 6) is used therapeutically as a class III antiarrhythmic agent for prophylaxis and treatment of ventricular arrhythmias that are unresponsive to conventional antiarrhythmic therapy. Like guanethidine, it is taken up and concentrated in adrenergic nerve terminals, initially releasing catecholamines and later, blocking release in response to neural stimulation. f^ 5 C H 3 0 " 3 CFJ3 > + - C H 2 N - C H 2 CH3CH2 \ 1 C H / 1 ,Br U Q Figure 6. Bretylium Figure 7. R1BA RIB A (radioiodinated bretylium analog), is an analog of bretylium which differs by replacement of the ethyl group on the quaternary nitrogen with a methyl group and 46 Laura N . A lco rn ,—' I Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y substitution of the aromatic ortho bromine with iodine (Figure 7). Animal studies have demonstrated localization of RIB A in both the heart and adrenal medulla. One of the first reports of using RIBA as a myocardial imaging agent is that of Counsell et al in 197397. Biodistribution studies in rats were done comparing R I B A to a bretylium analog containing a radioiodine in place of the bromo. Heart accumulation was much greater with RIBA and was thought to be due to rapid in vivo deiodination of the bretylium analog. In 1974, Counsell investigated RIBA for its usefulness as a myocardial imaging agent in rats and dogs1. Imaging in animals with and without myocardial infarcts demonstrated a dependency on iodine position in the aromatic ring . The ortho analog was more avidly taken up by the myocardium than the meta or para. Conversely, the para analog was more adrenospecific than the ortho. Also, R I B A was taken up less by infarcted heart tissue than normal heart tissue. A heart to blood ratio of 40, heart to lung ratio of 6.5, and adrenal to blood ratio of 13.8 were observed at 2 hours post-injection. In comparison, the heart/blood ratio has been less with tritiated M Q N B at 32.3 in the guinea pig and 31.5 in the rat6 7. Also, the adrenal/blood ratio has been as high as 30 with radioiodinated Q N B in the rat9 8. These studies were done using low specific activity material (approx. 851 kBq/mg). Significant uptake of RIBA in adrenal tissue was observed in another study in 9 9 dogs and again, iodine position was significant . However, localization in the adrenal gland was greater with ort/zo-iodinated analogs compared to para. This effect was less prominent with R I B A compared to the other N-substituted bretylium analogs that were 47 Laura N . A l c o r n ,—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y tested. The level in the adrenal gland was highest with RIB A at 2 hours post-injection and persisted throughout the 15 hours of study. In monkeys, humans and pigs, significant uptake of RIB A was found in the porcine myocardium, but no uptake occurred in primate hearts3. In four human volunteers, no evidence of cardiac uptake was found after imaging up to 24 hours post-injection. This was the first evidence that RIB A uptake by the heart was species dependent. In rats and dogs, Korn et al found RIBA was concentrated in the adrenal medulla 1 0 0. Like the work of Counsell et al discussed above, para-iodinated analogs had much greater uptake in the adrenal than ortho. The radiocarbon labeled analog was used to show that it was the quaternary form that was taken up by the adrenal gland and that high thyroidal activity after administration of the iodinated species was due to in vivo deiodination. Schreiber et al. investigated the mechanism of RIBA binding to rat cardiac tissue1 0 1. It was found to competitively displace the highly specific tritiated muscarinic antagonist 4 -NMPB (N-methyl-4-piperidyl benzilate) from homogenized rat heart tissue, with binding constants of 0 . 2 4 ± 0 . 1 2 p M and 0 . 9 7 ± 0 . 2 7 p M for the atrium and ventricle respectively. Subsequent studies by Gillard et al. indicated that bretylium also has partial agonist binding properties displaying preferential binding to muscarinic receptors labeled with the agonist [3H]oxotremorine M 1 0 2 . 48 Laura N . A l c o r n .—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y Thus, although initially promising in animals as a heart or adrenal imaging agent, primate and human studies have demonstrated that RIBA's biodistribution is species dependent and it appears to have no application in human medicine. 1.3.7.3. Dexetimide analogs. This is a compound with a unique structure (see Figure 8) compared to Q N B and RIBA. O Figure 8. lododexetimide In rats, tritium-labeled dexetimide was shown to bind to muscarinic receptors in the brain 1 0 3 . Uptake was stereospecific, saturable and displaceable. This was not observed with the pharmacologically inactive stereoisomer, levetimide. In another rat study, specific binding to rat heart was demonstrated in vivo by preinjection of atropine1 0 4. This was accompanied by a 63% decrease in accumulation of the radiotracer. Levetimide uptake was not affected by atropine treatment. Clearance from the blood was rapid with heart-to-blood ratios of up to 14:1. However, heart-to-lung and heart-to-liver ratios were less than one. In four epileptic patients, regional binding of iododexetimide was measured in vivo in the temporal lobes3 9. S P E C T imaging was used to compare 49 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical ^ Chapter 1. L I T E R A T U R E S U R V E Y ligand binding in the anterior hippocampus ipsilateral to the electrical focus compared to the contralateral hippocampus. A regionally specific change of receptor distribution in the anterior hippocampus in complex partial seizures of temporal lobe origin was demonstrated. Recently, Hicks et al. performed biodistribution studies in rats, rabbits and dogs with iodine-123 N-methyl-4-iododextemide105. Radioiodination was done using the chloramine T method and gave a radiochemical yield of 80%. The specific activity was greater than 70 GBq/pmol. High rat cardiac uptake (2.4% ID/g) was observed 10 minutes post injection, with a heart-to-lung ratio of 5:1. This is comparable to the ratio reported by Counsell of 6.5 with RIBA 1 . Hicks' blocking experiments with M Q N B and levetimide suggested specific receptor binding. In dogs, heart-to-lung ratios of greater than 2.5:1 at 10 and 30 minutes post injection were obtained. These were sufficient to permit S P E C T imaging. Higher activity was seen in the inferior wall of the heart compared to the anterior wall which the authors claimed was consistent with the parasympathetic innervation of the heart. Further research in primates or humans is needed to fully explore the role of this radiopharmaceutical for nuclear medicine imaging. 50 r Laura N . A l c o r n 1.3.7.4. Benztropine Figure 9. Benztropine [N- 1 1 Carbon-methyl]-benztropine has been investigated as a muscarinic receptor probe using P E T 4 5 1 0 6 1 0 7 . This anticholinergic agent also inhibits dopamine reuptake in the brain. In baboons, pretreatment with the potent dopaminergic antagonist N -methylspiroperidol reduced uptake of labeled benztropine in all brain regions. The greatest effect occurred in the corpus striatum, followed by the cortex, thalamus and cerebellum. This corresponds to the usual distribution of muscarinic receptor agents. In a second baboon study, preblocking with scopolamine and benztropine as well as the agonist pilocarpine also demonstrated specific binding to the muscarinic receptor. No effect on uptake in baboon brain was seen with pretreatment by the potent dopamine reuptake blocker, nomifensine. Kinetic studies showed marked differences in the rate of metabolism of the radioligand in baboons compared to humans. The metabolic rate in humans was much slower (83% unchanged ligand at 30 minutes post-injection versus 43%) in baboons). In 7 normal adult male volunteers (aged 19-82 yr.), m l and m2 51 Laura N . A lco rn ^ IL Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y receptor distribution was compared in the thalamic, hippocampal and cerebellar regions versus the frontal, parietal, temporal and occipital cortices as well as the corpus striatum. The authors felt their data suggested that regions high in m l and ml subtype density (the corpus striatum and the cortical mantle) showed a greater rate of receptor density decline with age than those areas with a low number of muscarinic receptors. Treatment of dementia in human patients using tacrine, a cholinesterase inhibitor has been monitored with P E T imaging using "C-benztropine5 2. Imaging of diseased brains showed little change in the distribution of this muscarinic agent compared to normal brains. However, radiopharmaceuticals used to image the nicotinic receptor, glucose metabolism and cerebral blood flow demonstrated improvements in brain function after treatment with these agents. 1.4 Chemistry of acylation and borane reduction reactions. For the synthesis of the two C T B A analogs, two acylation reactions and two borane reduction reactions were used. For this reason, the chemistry of these two reactions will be discussed in the following two sections. 1.4.1. Acyla t ion reactions. Acylation reactions involve the introduction of an acyl group (RCO-) in a compound. For the conversion of an amine to an amide, the reaction is usually one of nucleophilic substitution by the nitrogen at the carbonyl carbon and is represented by the following scheme: 52 Laura N . A lco rn ii Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y Scheme 2. Acylation Reaction^S R ' C O X + R 2 R 3 N H R ' C O N R 2 R 3 + H X The mechanism of the reaction is thought to be addition-elimination: Scheme 3. Mechanism of Acylation Reactions R C O X + Y " R - 6 -X l ki R C O Y + X In the case of amine acylation, loss of a proton from the nitrogen occurs as follows: Acy l halides are known to react readily and usually exothermically with secondary and primary amines to produce the amide. For this synthesis, further reduction of the amide to the amine was necessary. Another synthetic route to tertiary amines is via alkylation between an alkyl halide and the corresponding secondary amine 1 0 9. The reaction conditions used for acylation are generally dependent on both practical and thermodynamic considerations. For preparative syntheses, the availability, cost and safety of starting materials are often important factors in choosing the reagents. Scheme 4. Amine Acylation RlCOX + R 2 R 3 N H RCONR2R3 + X" 53 Laura N . A lco rn ^ 1 L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y For these reasons, the acid chlorides are frequently chosen. Other physico-chemical factors influencing the ability of an acid chloride to acylate an amine include: • the electron attracting properties of R in R C O X : CH3COCI > C H 3 C H 2 C 0 C 1 > C H 3 C H 2 C H 2 C 0 C 1 > ( C H 3 ) 2 C H C 0 C 1 C L C H 2 C O C l > P h C F L C O C l > C H 3 C O C l • electron withdrawing properties of X : I > Br > CI > F • nucleophilicity of amine (base strength) alkyl amines > aryl amines > amides A maximum yield of only 50% is possible due to consumption of the amine by the hydrogen halide released in the reaction leading to subsequent formation of the amine salt. A 50%) excess of the amine may be added to ensure efficient use of the starting acid chloride. Yield may also be increased by addition of inorganic or tertiary amine bases such as sodium hydroxide or potassium hydroxide. These exert a catalytic effect on the reaction, either by acting as proton acceptors or, with weakly nucleophilic amines, possibly form acylammonium salts of higher acylating power 1 0 8: Scheme 5. Formation of Acylammonium Salt R'COHal + R*N R'CON+R2 Hal-o 3 54 Laura N . A l c o r n 11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y For lower alkylamines and ammonia, the halide is usually added to a cold, stirred aqueous solution of the base. As the homologous series is ascended, yield has been found to decrease. This decreased yield may be related to formation of insoluble product around the unreacted halide and decreased contact between the hydrophobic acyl halide and water soluble amine. Yield may be increased by dissolution of the acyl halide in an inert solvent such as ether and vigorous agitation with the aqueous amine. A n immiscible solvent may be used and product is formed at the interface of the two phases. 1.4.2. Borane reduction Selective reduction of organic functional groups is often complicated by the presence of other, more labile groups within the molecule'1 0. The most common, broad-spectrum reducing agents are the metal hydrides and hydrogen (with a catalyst). Lithium aluminum hydride (L iAlH 4 ) is frequently used and is a powerful, nonselective reducing agent which was originally considered for this experiment as the agent to prepare both the secondary and tertiary amines from their respective amides. Scheme 6. Secondary amide reduction scheme o &-NHC(CH3)3 C H 2 NHC(CH3)3 Scheme 7. Tertiary amide reduction scheme. Crfe N-CtCHsh C=0 CH2N-C(CH^ 3 C H 2 55 Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y A problem with the use of this hydride can be unwanted attack of groups including the nitro, halogen, ester, sulfone and carbamate. Reductive cleavage of N -benzyl groups has occurred during L i A l H 4 reductions of benzamide derivatives. Extensive hydrogenolysis of the carbon-halogen bonds in both aromatic and aliphatic substrates is a potential problem and was of particular concern for this synthesis. The reaction mechanism for hydride reduction of tertiary amides involves the formation of a tetrahedral intermediate which may undergo both carbon-oxygen bond rupture leading to the amine, as well as carbon-nitrogen bond rupture to give the alcohol 1 1 1. This results in potential side reaction products which must be removed from the reaction mixture as well as compromising the yield of the desired product. Scheme 8. The reaction mechanism for hydride reduction of tertiary amides OM RCONR2' + MH RCNR2' H l e t r c h e d r a l i n te rmed ia te C-0 RCH 2NR 2' +M20 C - N cleovotje MH hydro lys is RCH2OM + MNR2' H20 R2'NH + RCHO • RCH2OH + R2'NH Replacement of some of the hydrogens of L i A l H 4 with alkoxy groups has resulted in a number of less reactive and more selective reagents. However, the borane reducing agents have been described as the reagents of choice for most reductions of carboxylic 56 Laura N . A l c o r n V - ^ I L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y amides to amines112""4. They have been effective as reducing agents for primary, secondary and particularly, tertiary amides (both aliphatic and aromatic)2. Reducing agents may be classified as either "basic", nucleophilic agents or "acidic", electrophilic agents114. The former class attacks electropositive atoms, i.e. centres of low electron density and the latter, electronegative atoms or centres of high electron density. Most of the metal hydrides are nucleophilic reagents and exert their action on the carbon atom of a carbon-hetero single or multiple bond. Conversely, borane is a Lewis acid or electrophile and attacks the hetero atom. This has implications on the rate and extent of the reduction reaction as well as resulting in a different pattern of selectivity for various functional groups. Nucleophilic reagents react only very slowly with the weakly electrophilic tertiary amide function and only very reactive members of this group such as lithium triethylhydroborate are effective. Thus, acyl halides are reduced very slowly by borane but carboxylic acids undergo reduction much faster than with lithium aluminum hydride. The advantages of diborane complexes as amide reducing agents may be summarized as follows: 1. Lewis acid character. 2. Low activation energies. 3. Clean reaction mixtures, minimum of side products. 4. Diborane is soluble in ether which results in homogeneous reactions, no induction period, and they are easily controlled. 5. The inorganic by-product of a borane reduction is usually an inert, water-soluble borate salt which can be washed away over a broad p H range. It is the unique chemistry of boron itself which results in the electron deficiency of its hydrides. It is the only group 3 A element that has almost completely non-metallic 57 Laura N . A l c o r n V-^ 1 1 L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y chemical and physical properties. It forms molecules whose bonding characteristics do not obey the normal laws governing valency and molecular structure. For example, diborane has fourteen valence shell atomic orbitals but only twelve valence electrons. With not enough electrons to permit the formation of two-electron bonds between all adjacent pairs of atoms, there is a resulting electron deficiency. This leads to multicentre bonding and the hydrides possess three centre, two electron bonds in the form of B—H--B bridges. H H H V V H ' V H Figure 10. Diborane Borane (BH 3 ) is the simplest of the boron hydrides and cannot be isolated. It exists only as a chemical intermediate. Diborane (B 2 H 6 ) is used as a source of borane and is volatile, reactive and oxidizes readily in air. Instant hydrolysis occurs with water. This reagent has found application in many reduction reactions involving alcohols, phenols, amines and thiols. The mechanism for the reduction reaction is thought to involve the formation of an adduct through attack by diborane on the carbonyl group" 2 1 1 4 . Diborane readily dissociates to borane which, acting as an electron pair acceptor forms coordination complexes with suitable electron donors. The intermediate, formed by electrophilic attack, is more stable and therefore long-lived, than the corresponding lithio intermediate. 58 Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y The borane-carbonyl complex rapidly rearranges to give the adduct. Subsequently the adduct undergoes hydride rearrangement and yields the amine via C - 0 bond cleavage. Scheme 9. Mechanism for Diborane Reduction RN RN + ? N C=0 • NC=0: BH 3 R' -C-OBH 2 R 1 ' R'y A RN • R ' - 6 -H + HoBOBH, l H Although diborane is available commercially in steel cylinders, the problems associated with diborane production and stability have resulted in the use of the borane-Lewis base complexes as a convenient source of borane during reduction reactions. Several complexes are available commercially. Both borane-tetrahydrofuran ( B H 3 - T H F ) and borane-dimethyl sulfide (BMS) have found usefulness in the reduction of amides. B H j - T H F results from a tetrahydrofuran solution of diborane. Its existence was predicted by the observation that the solubility of diborane in T H F was much greater than perfect solution predictions. The complex offers a convenient source of borane for reduction reactions. 0 : B H 3 Figure 11. Borane-THF Condensation of dimethyl sulfide and diborane on a vacuum line results in the borane-methylsulfide adduct: 59 Laura N . A l c o r n i=U Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y Scheme 10. Formation of the Borane-Methyl Sulphide Adduct 2(CH3)2S + B 2H 6 - (CH3)2S:BH3 This reagent has proved to be more convenient to use as a reducing agent than borane-THF, mostly because of its superior stability. The advantages of B M S over B H 3 -T H F are summarized as follows: 1. B H 3 - T H F is only available at a maximum concentration of 1M borane in T H F . B M S exists as the neat liquid with a borane concentration of ~10M. 2. Refrigeration of borane-THF is required to maintain hydride activity. B M S is more stable and may be kept for several months at room temperature and indefinitely in the refrigerator. 3. B H 3 - T H F contains less than 5mole% N a B H 4 to retard cleavage of the T H F . 4. B M S is soluble in and unreactive toward a wide variety of aprotic solvents. 5. Both reagents have similar reactivity although higher temperatures may be required with B M S . These are easily obtained since it is soluble in solvents such as toluene. The stoichiometry of the reduction reaction via B M S has been the topic of much experimentation 2' 1 1 3 1 1 5 1 1 9 . Primary amides undergo reduction by borane complexes used in stoichiometric amounts. Scheme 11. Reduction scheme for primary amides RCONH2 + 4/3 BH3.S(CH3)2 J K ~ R-CH2N(B^)2 MU. R-CH2NH2 60 Laura N . A l c o r n ii Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y Under the same reaction conditions, it was found that secondary and tertiary amides were -inert towards further hydride transfer reaction. In order to achieve complete reduction, five or six equivalents of hydride (an "active hydride" refers to one B - H bond) were needed to reduce tertiary or secondary amides, respectively. Scheme 12. Reduction scheme for secondary amides RCONHR' H ' B ' S W > . R-CH 2 NR , -B / • R-CH 2 NR'-B / - BH3 R-CH2NHR" Scheme 13. Reduction scheme for tertiary amides RCONR'R" 2 / 5 H ^ C H ^ RCH2-NR'R" RCH2-NR'R" • BH3 - M - R—CH2NR'R" Brown reported the use of boron trifluoride etherate to avoid the use of additional borane reagent to coordinate with the amine, forming as product the inert borane-amine adduct 1 1 3 1 1 5 . For secondary amides, six equivalents of hydride were recommended: one for evolution of hydrogen, two for the reduction and three to form the adduct. For tertiary amides, only five equivalents were required, since evolution of hydrogen does not occur. The complexed borane is wasted during purification of the amine product, making inefficient use of the hydride reagent. By carrying out the reaction in the presence of 61 Laura N . A lco rn IL Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y boron trifluoride etherate, the boron trifluoride combines preferentially with the amine since as a stronger Lewis base, it more avidly combines with the electrophile than the borane reduction reagent. Once the reaction is complete, the product can be isolated through addition of N , N , N ' , N'-tetramethlethylenediamine ( T M E D A ) , forming the insoluble addition compound with boron trifluoride. However, it has been found that the stoichiometry of the reaction can be controlled through simple temperature adjustments 1 1 9 1 2 0. This was confirmed in this synthesis and both the secondary and tertiary amide reductions were achieved with one mole of B M S in refluxing toluene. In this approach there is no need to distill off residual B M S and no acid hydrolysis is required for decomplexation of the amine-borane complex. 1.5 Statement of rationale and purpose The use of receptors for the early detection of disease by imaging assumes that receptor behavior reflects cell metabolic activity and function. It is also based on the fact that cell function usually becomes impaired before detectable changes in organ mass or morphology occur. Receptors should be particularly suited to this purpose since they represent a tissue-specific binding site for unique compounds and their density is thought to change in disease. Receptor density has been shown to both increase and decrease. Either scenario will permit detection by imaging, since identification of an abnormality is made by comparison to uptake in normal tissue. Interest in receptors as targets for radiolabeled imaging ligands has only recently developed. Much of the work has been done in cell preparations and animal models. However, in these systems investigators 62 Laura N . Alco rn r -^11L Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y have shown promising results with ligands directed towards the opiate91, dopaminergic 1 2 1 1 2 2 , adrenergic36 and both nicotinic and muscarinic cholinergic receptors53'65. For this project, the main area of interest is in the heart, where published work has been done primarily with adrenergic and cholinergic probes. These are the two main cell-surface receptors in this organ. They are both present in concentrations of 10 to 20 pmole/gram heart tissue70. The muscarinic receptor is of interest because compared to adrenergic and nicotinic sites, the m2 subtype population is very homogeneous. In fact outside the brain, this subtype is found in significant concentrations only in the heart, lungs, and uterus. Lung uptake may be anticipated to interfere with visualization of the heart but in humans the airways have a fourfold lower concentration of receptors. Even lower levels are found in the ileum. This is important from an imaging viewpoint since uptake in surrounding tissues and structures (background uptake) decreases the sensitivity of the scanning procedure. Although parasympathetic innervation of the ventricular myocardium is less than that of the atria, the presence of these fibers has been confirmed and found to be greater than was previously estimated65. In the cat, the concentration was sevenfold less than that of the atrium 1 2 3. The beta-adrenoceptor may be in greater concentration in the ventricles but possesses other disadvantages due to its high concentration in other surrounding non-target tissues including the lungs. The viability of this approach for assessing the early stages of cardiac disease is primarily hypothetical at this stage. The premise is that changes in the receptor population occur with pathology. This has been borne out by in vitro receptor binding 63 Laura N . Alco rn ur^-i IL Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y investigations as well as in vivo studies using animal and human subjects. There is also evidence in the literature that reactive oxygen metabolites which may be produced during ischemia affect binding of ligands to membrane receptors and coupling of receptors to G -proteins and effector enzymes5 1. It has been established that other nuclear medicine imaging procedures which depend on metabolic processes for uptake (e.g. bone scan), routinely show the presence of disease much earlier than procedures dependent on anatomical changes. Clinical work with IQNB, I M Q N B , Q N P and R I B A has indicated that accumulation in the myocardium may be sufficient to permit external imaging and may represent specific receptor uptake based on pre-blocking studies. Most of the work done to date in humans has been in patients with advanced disease. The myocardial uptake observed with the Q N B and RIBA analogues led to the synthesis of N-bis(o-iodobenzyl)-tert-butylamine (o-BIBTA), a compound which is a neutral tertiary amine and contains a similar tert butyl moiety as is found in R I B A 1 2 4 . The studies of receptor binding with RIBA demonstrated that the trimethylamine group gave superior myocardial uptake compared to the dimethylethyl amine group of the parent compound, bretylium. The work with Q N B demonstrated improved availability of labeled ligand for receptor binding if nonspecific binding to blood proteins was minimized by reducing the lipophilicity of the compound. One approach to this which resulted in a compound with high affinity for the receptor was replacement of the phenyl group with a cyclopentyl moiety9 0. This was therefore incorporated into the analog investigated in this project. The addition is analogous to the one in Q N B since they are both at the carbon alpha to the iodophenyl moiety. In preliminary work, an analog (o-64 Laura N . Alco rn i—11 Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y Figure 12. N-(iodobenzyl)(cyclopentylmethyl)-t-butylamine B I B T A ) not possessing the cyclopentyl moiety underwent biodistribution studies in mice with and without a preblocking dose of atropine1 2 4. High adrenal uptake which was significantly attenuated by the blocking compound was observed. The final structure for this research project is given in Figure 12. Both the ortho and para analogs were prepared and their biodistribution in mice was determined at various time intervals after intravenous injection. Additionally, a series of mice were studied following a pre-blocking dose of atropine in order to determine i f the distribution of the radiopharmaceutical was altered by the administration of this muscarinic antagonist. The hypothesis of this project is that a compound possessing the structural features of two previously established muscarinic radiopharmaceuticals and incorporating a series of modifications designed to (1) optimize binding to the m2 muscarinic receptor subtype and (2) improve localization in the myocardium on external imaging, may have usefulness in the early detection of cardiac disease. 65 Laura N . A l c o r n c=LI Muscarinic Radiopharmaceutical Chapter 1. L I T E R A T U R E S U R V E Y In order to obtain the preliminary data needed to evaluate the proposed analog, this research project undertook the following steps: 1. Synthesis of the ortho and para analogs of N-(iodobenzyl)(cyclopentylmethyl)-t-butylamine. 2. High specific activity radioiodination of the compounds with l 2 3 I. 3. Biodistribution studies in mice of the two radioiodinated analogs, with and without pre-blocking of the muscarinic receptors with atropine. 66 Laura N. A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS Chapter 2. SYNTHESIS 2.1 P r e l i m i n a r y research Dr. D. Lyster's lab began investigations into the development of a muscarinic receptor agent by incorporating the favorable structural attributes of R I B A and I Q N B 1 2 4 . Since R I B A is a quaternary amine, it may be predicted that significant penetration through physiological membranes may not occur. Also, R I B A studies have shown that substitution of the dimethylethyl moiety with a trimethyl group gives superior myocardial uptake results compared to its analogue. Therefore, a N-(orr/zo-iodobenzyl)-N-(benzyl) compound containing a similar tert-butyl moiety was designed and synthesis of this neutral tertiary amine was attempted. However, the synthesis resulted in an unexpected "dimerized" product: N-bis(o-iodobenzyl)-ferM5utylamine (o-BIBTA) (Figure 13). I Figure 13. o-BIBTA This compound shared some structural features with 3-quinuclidinyl 4-iodobenzilate (4-IQNB) since they are both tertiary amines with two bulky phenyl groups. As well, R I B A and o -BIBTA both possess an ortho iodo-benzyl moiety and tertiary butyl group. 1 2 3I-o-B I B T A biodistribution properties were studied in mice at one and two hours post 67 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS injection and are shown in Table 7. The uptake of l 2 3 I -o -BIBTA in selected organs using high and low specific activity preparations at one and two hours post-ingestion with and without atropine pre-blocking are given. Table 7. Biodistribution of ^ ^I-o-BIBTA at varying specific activities and with preinjection of atropine 60 min" 120min a 60min b 120 m i n b 60min/atropine c Blood 1.13±0.32 0.97±0.15 1.40±0.27 1.10±0.08 1.06±0.09 Adrenal 9 .58±1.44 17.23±2.23 61 .10±16.23 32.37±13.41 8.48±6.01 Heart 3 .50±1.38 3.19±0.44 5.91±0.67 4 .28±0.19 4 .36±0.75 Brain 0 .51±0.20 0.59±0.05 0.78±0.07 0.65±0.04 0 .58±0.07 "•b Represents the mean of five mice. Biodistribution o f 1 2 3 I -o -BIBTA(2 .69x l0 ' 1 0 moles per mouse). b Biodistribution o f 1 2 3 I -o -BIBTA(5 .59x l0 - "mole s per mouse). Represents the mean o f three mice, lmg /kg o f atropine was injected intramuscularly 30 minutes prior to 1 2 3 I - o - B I B T A (5.59xl0""moles per mouse) injection. ' , 2 3 I -o -BIBTA in both the high and low specific activity preparations showed significant uptake in the adrenal and heart tissues. The high specific activity material gave better target-to-nontarget ratios. Atropine pre-injection prior to administration of l 2 3 I -o -BIBTA was done to detect specific binding to muscarinic receptors. This decreased adrenal uptake by 86%. Heart uptake was less affected, decreasing by 26%. A Q N B analogue possessing a cyclopentyl moiety in place of one of the phenyl groups had improved binding affinity at ventricular muscarinic receptors compared to pulmonary receptors2'87. A n analog of o-BIBTA possessing such a group was thought to be worthwhile investigating as a potential myocardial localizing agent. Its structure is given in Figure 12, page 65. Based on this research and that of Gibson et al. with Q N B , the analog N -(iodobenzyl)(cyclopentylmethyl)-r-butylamine (ICTBA) was synthesized according to the following strategy. 68 Laura N . A l c o r n V - ^ I L Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS 2.2 Synthetic strategy 2.2.1 Reagents. Aldrich Chemical Co. Inc. (Milwaukee, Wis.): Toluene, anhydrous 99.8%, lot no. 03840BG; 2-(bromomethyl)tetrahydro-2H-pyran, 98%, lot no. 04712E2; 2-iodobenzoyl chloride, 98%, lot no. 04102HF, 10519DG; cyclopentane carbonyl chloride, 98%, lot no. 08520KZ, 10519DG; cyclopentane methanol, 98%, lot no. 00705AV; hexamethyl ditin, 99%, lot no. 03803TT; tetrakis(triphenylphosphine)-palladium (0), 99%, lot no. 01101CY, 01816PF; bis(tributyltin), 97%, lot no. 00720EY; 1,3dicyclohexyl carbodiimide, 99%>, lot no. 08528DZ; borane T H F (borane-tetrahydrofuran complex), lot no. 00214CG; iodine, 99.8%, lot no. 06306TY; 2-iodobenzylalchohol, lot no. 09818EW; sodium, lump, in kerosene, 99%, lot no. 08002CW. Lancaster Co., Eastgate, England: 4-iodobenzoyl chloride, 98%, lot no. 19128; borane dimethyl sulphide complex, lot no. 09001; N-benzyl-tert-butylamine, lot no. P04362. B D H Inc., Toronto, Ont.: silica gel 60, 70-230 mesh; concentrated sulfuric acid, lot no. 114172/30094; tetrahydrofuran, lot no. 34346449; benzophenone, lot no. 5004180H; sodium nitrite, lot no. 89245; sodium sulfite, lot no. 105015; sodium sulphate, lot no. 112446. > Fisher Scientific, Nepean, Ontario: magnesium sulfate, anhydrous, lot no. 930992B; sodium hydroxide; hydrogen bromide, lot no. 790270; all solvents unless otherwise specified. 69 Laura N . A l c o r n ^ L l L Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS Absolute ethanol was obtained from Stanchem Inc., Vancouver, B.C.. 7er/-butylamine, lot no. 6772 was obtained from Eastman Organic Chemicals, Rochester NY. Hexamethylditin, 95%, lot no. A30F28 was obtained from Alfa, Johnson Matthew, Wardhill, M A . Sodium bicarbonate, lot no. 50H0135 was obtained from Sigma Chemical Co., St. Louis, M O . Concentrated hydrochloric acid, lot no. 0713353, was obtained from Allied Chemical Canada Ltd., Pointe Claire, Que. Lithium bromide, anhydrous, lot no. 0010165, was obtained from Matheson Coleman and Bell, Norwood, Ohio. Fluothane® (halothane BP), lot IMFJ-E2, exp. 93NO was obtained from Ayerst Laboratories. Atropine 0.6mg/ml, lot 10520NJ, exp Nov. 1.99 was obtained from Abbott Laboratories, Montreal, Quebec. [ , 2 3I]NaI in 0.1 N N a O H (39 M B q / u L ) , various lots, was obtained from Nordion International, Vancouver, B.C. 2.2.2. Equipment, instrumentation. PolyPak™ Cartridges, Glen Research, Sterling V A ; Baker-flex™ silica gel, IB2-F, J.T. Baker Inc., Phillipsburg, NJ; Whatman K C 1 8 F 200 u M Reverse Phase T L C plates, 2.5 x 6.7cm, Whatman Int., Ltd., Maidstone, England, Capintec™ Radioisotope Calibrator C R C - 5 , Capintec Inc., Montvale NJ; Mineralight UVGL-58 Lamp, U V 254/366 N M , UVP Chromato-vue Cabinet Model 00-10, San Gabriel, C A ; Buchi Rotavapor-R™, Flawil, Switzerland; H P L C gradient controller and pump, Waters (Waters Chromatography Div., Milford, M A ) , model 510; H P L C U V detector, Waters 740 Data Module, H P L C injector, Waters model U6K; H P L C Radioactivity Detector, B B C Goertz Metruwatt, SE120; C-18 5p Ultremex Semi-prep column, 250x10mm, serial # SP/7376A, Phenomenex®, Torrance, C A . . Bomem MB-100 Fourier transform 70 Laura N . Alco rn ,—11 Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS spectrophotometer, Bomem Inc., Quebec, Canada. Pye Unicam SP1000 Infrared Spectrophotometer, Cambridge, U K . Counting tubes for the biodistribution experiments were 12x75mm culture tubes, PS Lot 4181036696, Simport Plastics Ltd., Quebec, Canada. Organ weights were obtained on a Sartorius Listed Scale, model B120S-OKR "basic", Sartorius G M B H , Gdttingen, Germany. Samples were counted on a Cobra II, Well Gamma Counter, Packard Instrument Co., Meriden, C T , U S A . 2.2.3 Synthesis. 2.2.3.1. Acylation of 2 or 4-iodobenzoyl chloride (formation of o-or p-iodobenzyl-tert-butylamide): IBTAmide. To a solution of ter/-butyl amine (3.5 ml, 33.4 mmol) in 50:50 acetone in water (38 ml) in a 100 ml round bottom flask, was added 2- or 4-iodobenzoyl chloride (4.0 g, 15.0 mmol) with stirring. Heat was evolved with the addition. The p H was adjusted to 9.0 if necessary with 1 M N a 2 C 0 3 . The mixture was stirred for 2 hours, adjusting the p H as needed. The reaction was neutralized with 1 M HC1 to pH 7.0. Cooling at 4°C for 2 hours to cause crystallization followed. The mixture was filtered and washed with cold water. The crude product was recrystallized from 50:50 ethanol in water. The final product was white crystals. Formula C n H 1 4 N I O , molecular weight 303.1 g/mol. 2.2.3.2. Borane reduction of IBTAmide (formation of o- or p-iodobenzyl-tert-butylamine): IBTAmine. A solution of o- or ;?-iodobenzyl-terr-butylamide (3.03 g, 10 mmol) in 10ml anhydrous toluene was stirred on ice under anhydrous conditions. Borane methyl sulfide (1.05 ml, 10.5 mmol) was added dropwise and the mixture was stirred at 0°C for 15 minutes. Refluxing followed for 2 -4 hr. until product was identified by T L C analysis. Aqueous 71 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS N a H C O j (10%, 15 ml) was added and the mixture was stirred for 30 minutes. The aqueous layer was separated and extracted with ether. The combined organic fractions were added to the toluene layer and dried over magnesium sulfate. The solvent was removed under reduced pressure and the residue distilled in vacuum using a Kugelrohr apparatus to give the product as an oil. Formula C U H 1 6 N I , molecular weight 289.9 g/mol. 2.2.3.3. Acylation of IBTAmine (formation ofN-(iodobenzyl)(cyclopentylmethyl)-t-butylamide): ICTBAmide To a solution of methylene chloride (4 ml) and 10% N a O H ( a q ) (4 ml) was added o- or p-iodobenzyl-terr-butylamine (373 p L , 500 mg, 1.73 mmol) with stirring. To this mixture was added dropwise cyclopentanoyl carbonyl chloride (345 mg, 2.595 mmol). The mixture was stirred vigorously for 24 hours and the aqueous and organic layers separated. The aqueous layer was extracted with methylene chloride and all organic fractions were combined and washed with 1 N HC1 and water. The organic fractions were dried over magnesium sulfate and the solvent was removed under reduced pressure. The crude product was recrystallized from 70% aqueous ethanol in the case of the ortho and was distilled to a colorless oil with the para. Formula C 1 7 H 2 4 N I O , molecular weight 385.3 g/mol. 2.2.3.4. Borane reduction of pentylated amide (formation ofN-(iodobenzyl)(cyclopentylmethyl)-t-butylamine): ICBTA A solution of N-(iodobenzyl)(cyclopentylmethyl)-t-butylamide (100 mg, 260 pmol) in 2 ml anhydrous toluene was stirred over ice under anhydrous conditions. Borane methyl sulfide (27 u L , 270 pmol) was added dropwise and the mixture stirred at 0°C for 15 minutes. Refluxing followed for 2 - 4 hours or until product was identified by T L C 72 Laura N . A lco rn c=U L Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS analysis. The mixture was cooled to room temperature and 10% N a H C 0 3 ( a q ) (390 pL) was added. The aqueous layer was extracted with ether. A l l organic layers were pooled and dried with anhydrous magnesium sulfate. The solvent was removed under reduced pressure and the crude oil was distilled in a Kugelrohr apparatus. Formula C , 7 H 2 6 N I , molecular weight 371.1 g/mol. 2.2.3.5. Radioiodination [123I]NaI in 0.1 N N a O H (39 M B q / p L ) was obtained via the 1 2 4Xe(p,2p) 1 2 3I reaction (Nordion International). Isotopic purity was 123I>99.8%, iodide >99.0%. A mixture of cupric sulfate (15 mg, 60 pmol), L-ascorbic acid (1000 mg, 5.68mmol) and tin (II) sulfate (50 mg, 233 pmol) was prepared under anhydrous conditions. Radiolabeling kits were prepared in advance by dispensing 10 mg aliquots of the mixture into dried borosilicate serum vials fitted with rubber stopper and aluminum crimp. Each kit contained ascorbic acid (10 mg), C u S 0 4 • 5 H 2 0 ( 150 pg), and SnS0 4 (500 pg) and was kept under nitrogen atmosphere. The kits were used within 48 hours of preparation. To one kit was added 1 ml 0.01 M H 2 S 0 4 and the vial was agitated until dissolution was observed. The contents were transferred by plastic syringe (1 mL) fitted with a hypodermic needle (2" x 24 G A ) to [123I]NaI (9 uL, 0.3 - 0.4 GBq). This mixture was drawn "up similarly and transferred to a 5 ml serum vial containing 1000 pg of the iodo-analog under nitrogen. The vial was placed in a heating block at 85 °C for 30 minutes. The ortho analog was further purified by passage through a Styrene CI8 SepPak. 73 Laura N . A l c o r n Ur^ I L Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS 2.2.4 Identity testing. The ortho intermediates and product underwent more extensive identity testing than the para because this analog was synthesized in its entirety prior to attempting synthesis of the para. Since the two species have very similar structures, the use of less expensive and lengthy analyses especially for the intermediates, was considered adequate to confirm the presence of the para species. Both normal and reverse phase thin layer chromatography were performed on "mini" (2.5 x 6.7cm) T L C plates. The method was used to monitor disappearance of reactants and appearance of products during each synthetic step. It was also used to determine radiolabeling efficiency. Eluting conditions were optimized to provide maximal separation between reactants and products or between bound and free radioiodine. Normal phase chromatography separates solutes based on their adsorption properties via hydrogen bonding. It is usually done with compounds which are soluble in organic solvents and are not ionized. Semi-polar solvents such as isopropanol and chloroform are usually used. Blending is frequently helpful in achieving the desired polarity. It was useful for separating the precursors and side products from the end-products for most of the intermediates and final analogs. Some problems with streaking were encountered, probably due to ionization of the secondary and tertiary amines. The system was also useful for determining radiolabeling efficiency. Free iodine remained at the origin and the analog migrated with the developing medium. The systems used for each compound are given in Table 10 and Table 11, pages 88 and 89. 74 Laura N . A lco rn V - ^ I L Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS Reverse phase chromatography uses partition mechanisms to separate compounds. A non-polar to semi-polar stationary phase is bonded onto a support phase which is usually silica. A n example of a non-polar stationary phase is octadecyl silane (ODS) which is a C-18 hydrocarbon. Blending is often used to fine tune the elution volume. This approach was required to fully discriminate the amide from the amine compound due to problems with ionization and streaking in the normal phase systems. *H N M R was performed in the N M R Laboratory of the Faculty of Chemistry, University of British Columbia (UBC) using a Bruker AC-200 instrument (Bruker Instruments Inc., Billerica, M A ) at 200 or 400 M H z and expressed as parts per million (a) from the internal reference tetramethylsilane. The compounds were dissolved in various deuterated solvents as specified in Table 10 and Table 11, pages 88 and 89. The sample was dried overnight under vacuum. For oils, 3 mg was dissolved in 1 ml of solvent, filtered and sealed in an N M R tube. For powders, 5 mg was dissolved. This was used to obtain structural information for the purified intermediates and to confirm that the desired product was synthesized. Elemental analysis was performed by Canadian Microanalytical Services, Delta, B . C . A 2-3 mg sample was dried overnight and sealed in a glass tube prior to shipping. This analysis also provided identity information but was used only when there was a discrepancy between the results of two other analytical methods or to provide definitive evidence that the final I C T B A products were synthesized. 75 Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS H P L C was used as a purification method for preparation of samples needed for identity testing or as a potential method for separation of bound from free iodine. It was performed on a Waters system as described in Section 2.2.2. (Equipment, instrumentation) page 70 using the following parameters: Column: Ultremex 5 p. C 18 Dimensions: 250 x 10 mm Mobile phase: 95% absolute ethanol, 5% 2.0M triethylamine acetate (285 ml ethanol + 15 ml 2 M TEA/acet.) Flow rate: 2 ml/min Pressure: 1500- 1600 psi Temperature: ambient Detector: U V @ 254 nm, radioactivity detector, set empirically for maximal count rate Sample size: 50 pg/50 p L elution medium or labeling reaction mixture Triethylamine acetate was added to the medium in order to prevent binding of the analog to the silanol residues on the column. This had been used previously with M I B G (meta-iodobenzylguanidine). Primary amines were removed from the T E A by refluxing over tosyl chloride. The acetate salt was formed by preparing a 2 M triethylamine solution in acetic acid. This reagent was also useful since it provides a low U V background. 76 Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS Mass spectrometry (MS) was performed either by Dr. Frank Abbott's laboratory in the Faculty of Pharmaceutical Sciences at U B C or by the U B C Mass spectrometry Laboratory, Faculty of Chemistry, under the supervision of Dr. G . Eigendorf. This technique was used to obtain molecular weight information. In Dr. Abbott's lab a Hewlett Packard Model 1090 Series II Liquid Chromatograph (Hewlett Packard, Avondale, PA) coupled to a V G Quattro Quadrupole M S / M S (Fisons, Altrincham, England) system was operated in the atmospheric pressure chemical ionization (APCI) or electrospray (ES) modes. During scan mode, M S conditions were: Parameter Electrospray Atmospheric Pressure Chemical Ionization Source temperature: 100°C 100° C Cone voltage: 20-30 V 25 V Flow rate: 1 ml/min 1 ml/min Sample: I B T A m i d e and IBTAmine : l O p p m i n o - I C T B A : 20 ppm in methanol (10 y.L 50:50 MeOH:water o f a solution o f 2 mg/ml in C H 2 C 1 2 in 1 ml M e O H ) o - I C T B A : 10 ppm in methanol and aq. Formic acid (10 nL o f a solution of 2 mg/ml in C H 2 C 1 2 in 1 ml M e O H ) , added in equal parts to a mixture o f 50% water + 0.1% formic acid Scanning speed: 100 amu/sec lOOamu/sec To facilitate ionization, an equal amount of 50% water + 0.1% formic acid was added to the sample of o - ICTBA used for ES. This increases the sensitivity for positively charged ions by promoting formation of H+adducts. High resolution mass spectrometry was done on a Kratos M S 50 (Kratos Analytical, Ramsey, NJ) using electron impact mode at 70 eV. This was used in the final 77 Laura N . A lco rn i—' 1 Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS identity testing of the I C T B A analogs due to conflicting and variable results obtained from elemental analysis. IR spectroscopy of the compounds was done initially to confirm the presence of the amine function in o-IBTAmine, after the reduction step with o-IBTAmide. On the N M R spectra, the nitrogen associated proton was usually not visualized. Samples were prepared by trituration to a fine powder with one or two drops of Nujol using an agate pestle and mortar. A Pye Unicam SP1000 Infrared Spectrophotometer was used for these intermediates. For completeness, FTIR was also done with o - ICTBA to provide a "fingerprint" identification method for the final product. It was not performed on the para analog. Samples were prepared using the neat oil on halide plates and analyzed using a Bomem MB-100 Fourier transform spectrophotometer (Bomem Inc., Quebec, Canada) using Bomem Easy Version 1.50B FTIR software (Hartmann & Braun) . 2.3 Results and discussion 2.3.1 Synthesis The overall synthesis consisted of four steps followed by radioiodination. These steps were the same for both analogs although the reaction conditions varied slightly for each. The synthetic steps for the ortho analog are given in Scheme 14. Steps one and three were both acylation reactions and steps two and four were borane reduction reactions. The final step was an exchange labeling reaction in which 1 2 3I was added either to the ortho or para position of the final tertiary amines. 78 Laura N . A l c o r n i=L! Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS For the synthesis of the two amines, two alkylation reactions between the appropriate alkyl halide and corresponding amine would have provided a more direct route to the final products. However, this was not successful with either synthesis. Some disadvantages of the alkylation approach include poor yield and side-product formation, resulting in the need for more complex purification steps. This was found in preliminary syntheses of o-IBTAmine from 2-iodobenzyl bromide and tertiary butyl amine. Also, problems were encountered with salt formation of both o-IBTAmine hydrochloride and the hydrobromide salt of tertiary butyl amine. In the case of o -ICTBA, no evidence of product was observed with reaction of cyclopentane methyl bromide and o-IBTAmine, possibly due to inadequate nucleophilicity of the starting secondary amine. This was compounded by the lack of commercial availability of an alkyl halide form of the cyclopentyl group and difficulties with synthesizing the methylbromide species from the methanol analog. A cleaner, more efficient strategy was to use a two-step synthesis involving production of the amide from the acyl halide and primary or secondary amine followed by reduction to the desired amine. The disadvantage of this approach was the need for the additional reduction step. Steps 1 and 3 in Scheme 14 show the two acylation reactions. Yields of approximately 95% were obtained for o-IBTAmide synthesis and 86% for /7-IBTAmide. For the acylation reaction between ort/20-IBTAmine and cyclopentane carbonyl chloride, the yield was not as high at 62%. The decreased yield may be 79 Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS Scheme 14. Synthesis of o-ICTBA Stepl. NH2C(CH3)3 p H 9 N H C ( C H 3 ) 3 Step 2. < ^ ^ - ? - N H C ( C H 3 ) 3 BMS ( ^ ^ - C H 2 N H C ( C H 3 ) 3 Step 3. O, ,CI < ^ - C H 2 N H C ( C H 3 ) 3 + £j] 1 0 c ^ ^ < ^ - C H 2 N C ( C H 3 ) 3 Step 4. \0/ C H 2 N C ( C H 3 ) 3 ^ — ' 6=0 B M S / Q V c H 2 N C ( C H 3 ) 3 N ' Q H 2 related to formation of insoluble product around the unreacted halide and decreased contact between the hydrophobic acyl halide and water soluble amine. Also, difficulties with precipitation of the salt form of ort/zo-IBTAmine during the reaction resulted in the need for a two-phase system of toluene and aqueous sodium bicarbonate. Presumably the protonated nitrogen prevented attack by the electrophilic carbonyl carbon of cyclopentane carbonyl chloride. The alkaline aqueous phase kept the amine in the form of the base. 80 Laura N . A l c o r n V-^ i L Muscarinic Radiopharmaceutical Chapters SYNTHESIS Salt formation was not observed with the para analog and the yield with the acylation reaction for this analog was 85%. Lithium aluminum hydride was initially considered for the reduction reactions of step 2 and 4. This approach however has caused hydrogenolysis of carbon-halogen bonds in aromatic compounds. Borane reduction reactions have been used with good yield and little need for removal of side products in the formation of amines. Initially in this synthesis, B H 3 - T H F was tried as the reducing agent but subsequently B M S was found to produce superior yields and was associated with a simpler, faster procedure. Borane reduction from IBTAmide to IBTAmine was achieved with a yield of 80%) with both analogs. The second borane reduction gave a yield of approximately 85% with the ortho species and 60% with the para. The overall yield for the synthesis of o -ICTBA was 39% and for p-lCTBA was 37%. 2.3.2 Identity Testing and Radiochemical Analysis Not all intermediates underwent all analyses. See Table 8 for a summary of the analytical techniques applied for the precursors and products. See Appendix 1. Spectral Analysis., page 112 for the spectra of the final I C T B A products. 81 Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS Table 8. Analytical techniques Used in Identity Testing for ICTBA and precursors IBTAmide IBTAmine ICTBAmide ICTBAmine Analysis ortho para ortho para ortho para ortho para TLC, Reverse and normal phase .V 111111 • V , - y : v : V. SSlllIllllIl NMR : ; m f s 4 v V Elemental Analysis V W illlllli HPLC R l . U V U V , Ra U V , R a Mass spectrometry KS(T). E S ( - ) ES(+) , E S ( - ) APCI. ES(-t), H i Res H i Res FTIR V V T L C = thin layer chromatography: U V = ultraviolet detection: RI = refractive index detection: E S ( + ) = electrospray; positive ion mode: E S ( - ) = electrospray; negative ion mode: R a = radioactivity detection: A P C I = atmospheric pressure chemical ionization mode: H i Res = high resolution mode (electron impact) 2.3.2.1. T L C , N M R and E L E M E N T A L A N A L Y S I S The results of T L C , elemental analysis and N M R analysis for the intermediates and final products are given in Table 10, page 88 for o - ICTBA and Table 11, page 89 for J 9 - I C T B A . N M R and T L C were the two main techniques used to identify reaction products. This was primarily due to their easy availability and relative low cost. The mass, IR, and N M R proton spectra as well as the chromatograms from H P L C analysis for the intermediates in the synthesis of both I C T B A analogs are not provided. The chemical shifts from N M R spectrometry of the intermediates are given in Table 10 and Table 11. 82 Laura N . A l c o r n .—11 Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS The proton spectra for o - ICTBA and p - I C T B A are given in Figure 23 and Figure 24 , page 112. As expected, the spectra are very similar for the two analogs due to their related chemical structures. For both, all peaks integrated to the expected number of protons. A singlet is present in both spectra at a = 7.27 and is due to the trace amounts of protonated chloroform which is present as a contaminant in the deuterated species, used as the solvent for both analogs. Similarly, a singlet at a = 1.55 is due to trace amounts of water in the sample despite efforts to use anhydrous materials and equipment. The large singlet due to the nine protons of the tertiary butyl group can be seen at a 1.04 ppm for o - ICTBA and 1.00 ppm for p - I C T B A . This is within the expected range for the proton chemical shift of a tertiary butyl group 1 2 5 1 2 6 . Immediately downfield to this singlet is a series of at least three multiplets in the region of a = 1.2 to 1.70 ppm which integrate to the number of protons in the pentyl group. By comparison to the spectra for cyclopentane carbonyl chloride and cyclopentane this is the expected region for the aliphatic protons of this moiety 1 2 6. A n attempt was not made to assign the signals within each multiplet to the corresponding protons. A doublet is evident at a = 2.47 ppm for the ortho and a = 2.39 ppm for the para C T B A analogs and represents absorption by the protons of the methylene group alpha to the nitrogen and pentyl groups. Since there are two, integration provides two equivalents, and since they are coupled to a neighboring carbon which is bound to one proton, the signal is split and given as a doublet. The singlet at a = 3.6 ppm is due to the absorption of the two protons of the methylene group alpha to the aromatic ring and is not split since no neighboring atoms are bound to protons. The identity of the protons responsible for this signal was partly determined 83 Laura N . A l c o r n ,=11 Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS through comparison to the spectra of compounds containing similar linkages. However, the available reference spectra were for compounds with unsubstituted aromatic rings only. The iodine species caused deshielding of the protons from the region of a = 3.5 in N-(ter/-)Butylbenzylamine, and N-butylbenzylamine to a = 3.6 with the C T B A analogs 1 2 5 1 2 6 . I Figure 14. Ortho- ICTBA The aromatic protons absorb in the region of a = 7 ppm and it is here that the spectra for the two analogs differ. The signals in this region for o - ICTBA include a pair of triplets at a = 6.87 ppm and 7.26 ppm. The splitting pattern indicates these are due to the para and meta (3 and 4 position) protons. The deshielding effect of iodine would likely cause the triplet of the3-osition proton to be more downfield than that of the 4-position The doublets at 7.72 ppm and 7.79 ppm are caused by the ortho and meta (2 and 5 position) protons. Again, the deshielding effect of the iodine would be expected to cause the 2-position proton to be more downfield than the 5-position. 84 Laura N . A l c o r n uyjjL Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS o Figure 15. Para-ICTBA In the spectrum obtained for p - I C T B A , the aryl hydrogens are represented by the two sets of doublets in the region of a = 7.7 ppm. Only two signals are obtained for the four protons since the chemical environment of the two ortho protons is identical to that of the two meta protons. They are both doublets since all protons have a neighboring carbon bound to a single proton. As expected, both doublets integrate to two equivalents. The deshielding effect of the para iodine on these aryl hydrogens would be expected to cause the two meta hydrogens to be downfield in relation to the ortho pair. Results from elemental analysis are within 5% of expected values. However, considerable variation in results were obtained and this led to confirmatory testing using high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. The spectral results from H P L C , mass spectrometry and infrared spectroscopy are given in Appendix 1. Spectral Analysis., page 112. 85 Laura N . A l c o r n li^U L Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS 2.3.2.2. H P L C The H P L C chromatograms are provided only for the final radioiodinated products. Both U V absorption and radioactivity detection were used. Figure 25 and Figure 26 (Appendix 1. Spectral Analysis., page 112) show the ultraviolet and radioactivity chromatograms following H P L C of o-ICTBA. Figure 27 and Figure 28 (page 114) give the corresponding recordings for p - I C T B A . Note that the U V absorbance is charted approximately 0.2 minutes before radioactivity. The radioactivity recordings were made following injection of the reaction mixture. However for demonstration purposes, the U V recordings are given for the pure analog. The equivalent U V recordings for the reaction mixtures contained peaks contributed by the other reactants which in the case of ascorbic was very large compared to the signal from the Table 9. HPLC Retention Times for o-ICTBA, p-ICTBA and Free Iodine Analog Retention Time (minutes) , 2 3 I o - I C T B A 9756 1 2 3 I p - I C T B A 9.61 free iodine 6.0 - 8.0 ligand. This made the ligand's peak more difficult to resolve. Table 9 gives the retention times for the radioiodinated I C T B A species and free iodine. Good separation was obtained between free and bound radioiodine. This vould have permitted a method for purifying the final radioiodinated product if there was an unacceptably high level of free radioiodine. A disadvantage with this approach for removal of free radioiodine is the need for multiple injections of the reaction mixture in order to collect sufficient quantities 86 Laura N . A l c o r n V-^ IL Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS of radiolabeled material. However, this proved to be unnecessary due to acceptable radiolabeling efficiencies with both I C T B A analogs. 2.3.2.3. Mass spectrometry Mass spectrometry was used to obtain molecular weight information. Soft ionization processes are preferred for this purpose since good visualization of a molecular ion peak is usually possible. Atmospheric pressure chemical ionization (APCI) or electrospray (ES) were tried with both I C T B A products. These offer several advantages for molecular ion production. Electrospray can be used at relatively low temperatures and molecular ions are frequently produced with little fragmentation. It is most suited to polar species. APCI has most of the advantages of ES but thermal degradation can be a problem. This is due to the need for heating the sample (probe temperatures ~ 300 °C) to produce a vapor. Ionization of the solvent (usually methanol) results in the species responsible for proton transfer. It may be used for compounds which do not readily ionize. Other features include suitability for aqueous phases, low maintenance and ability to accommodate normal L C flows without splitting. Figure 29 and Figure 30 (page 115) show the mass spectra for o - ICTBA (371.1 g/mol) using APCI and ES , respectively. A strong pseudomolecular ion peak corresponding to the protonated species [MH + ] , was obtained with both interfaces at m/z 372. The pseudomolecular peak and second peak at m/z 316 on the ES spectrum are both off-scale. The peak at m/z 316 [M-55] is 87 Laura N . A l c o r n V-^ IL Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS presumably due to cleavage of the tertiary butyl group [m/z 57] and possibly addition of a proton in place of the terr-butyl followed by protonation of this species to produce the cation. At a lower cone voltage (20 V) this peak became much less prominent and the molecular peak remained as the base peak (spectrum not provided). The [M-55] peak was also visualized on the APCI spectrum less intensely (approximately 10% abundance). The M H + +1 peaks at m/z 373 are likely a result of the natural isotopic contribution from carbon 13 which would be expected at a relative abundance of 19%) in a 17-carbon Table 10. Physico-Chemical Properties of the Intermediates and Final Product for o-ICTBA Parameter IBTAmide IBTAmine IBTAmine pentylated pentylated base salt IBTAmide (ICBTAmide) IBTAmine (ICTBA) formula C u H l 4 N I O C M H ^ N I C n H 1 6 N I H C l C l 7 H 2 4 N I O C , 7 H 2 6 N I mass 303 289.9 327.9 385.29 371.11 appearance white crystals colorless oi l not observed colorless o i l colorless o i l Rf on TLC, normal phase 70:30 hexane: ethylacetate 0.60 0.50 - 0.60 (streaks) 0.70 0.89 Rf on TLC, reverse phase 100% methanol 0.71 0.72 0.57 0.55 NMR features,1H (solvent) a 1.20 (s,t-but), a 5.80 (s, N-H), a 7.40 (d, m-arH's), a 7.70 (d, o-arH's) (CDC1,) a 1 10(s,t-but), a 3.60 (s, N-CH2), a 7.10 (d, m-arH);a 7 60 (d, o-arH's) (CDCI3) a 1.40 (s,t-but),a 1 60 (m.pentyl-H'S),CT2.60 (p, 1-Cpcntyl), a 4 50 (s,N-CH 2 )a7.0 (d, m-arH's), a 7.6 (d, o-arH's) (CDC1,) a 1.0 (s,t-but), a 1.20 (m,pentyl-H's), a 1.40 (m,pentyl-H's), a 1.70 (m.pentyl-H's), a 2.39 (d, N, pentyl-CH2), a 3.60 (s, N-CH 2 ) ,a 7.15 (d, m-arH's), a 7.55 (d, o-arH's) (CDC1,) elemental analysis: % C, H, N calculated: 43.6, 4.7, 4.6 45.7, 5.5,4.8 40.6,5.2,4.3 53.0, 6.3,3.6 55.0 ,7 .1 ,3 .8 found: n/d n/d n/d n/d 55.0 ,6 .98,4 .0 n/d = not done 88 Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS compound. This abundance cannot be confirmed in Figure 30 due to the base peak being offscale. Table 11. Physico-Chemical Properties of the Intermediates and Final Product for p-ICTBA Parameter IBTAmide IBTAmine IBTAmine pentylated pentylated base salt IBTAmide (ICTBAmide) IBTAmine (ICTBA) formula C u H l 4 N I O C „ H 1 6 N I C „ H 1 6 N I H C 1 C 1 7 H 2 4 N I O C 1 7 H 2 6 N I mass 303.1 289.9 327.9 385.3 371.1 appearance white crystalline needles colorless o i l white cuboid crystals white, shiny flakes colorless o i l 1 Rf on TLC, normal phase 70:30 hexane: ethylacetate .38 . 50 - .55 (streaks) origin .73 .90 Rf on TLC, reverse phase 100% methanol .75 . 5 0 - .80 (streaks) .48 .54 NMR features,1H (solvent) CT 1.28 (s,t-but), CT 5.50 (s, N-H), a 7.0 (t, m-arH), CT 7.30 (d&t, o&p-arH's), a 7.80 (d, m-arH) (CDC13) CT 1.20 (s,t-but), CT 3.70 (s, N-CH2), CT 6 9 (t, m-arH), CT 7.25 (t, p-arH), CT 7.40 (d, m-arH), CT " 7.72 (d, m-arH) (CDC1,) CT 1.40 (s,t-but), CT 4.20 (s, N-CH 2), CT 7.2 (t, m-arH), CT 7.5 (t, p-arH), CT 7.0 (2 d's, o&m-arH's), CT 9.2 (s, N-H) (DMSO) CT 1.40(s,t-but),a 1.70 (m.pentyl-H'S),CT2.50 (p, 1-Cpcnlvl). CT4 25 (s.N-Cll;, CT7 0(1, p-arH), CT 7.2 (d, m-arH), a 7.4 (t, m-arH), CT 7.8 (d, o-arll) (CDCI,) -CT 1.04 (s,t-but), CT 1.23 (m,pentyl-H'S),CT 1.42 (m,pentyl-H's), CT 1.62 (m.pentyl-H's), CT 1.70 (m,pentyl-H's), CT 2.47 (d, N, pentyl-CH 2), CT 3.60 (s, N-CH 2), CT 6.87 (t, p-arH), CT 7.26 (t, m-arH), CT 7.72 (d, 0-arH),CT7.79 (d, m-arH) (CDCI,) elemental analysis: % C, H, N calculated: 43.6, 4.7,4.6 45.7,5.5,4.8 40.6, 5.2, 4.3 53.0, 6.3,3.6 55.0 ,7 .1 ,3 .8 found: n/d 46.1,5.6, 4.8 n/d n/d 54.9, 7.0, 3.9 n/d = not done 89 Laura N . A l c o r n ,=0 Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS A small peak at m/z 217 on the ES spectrum may be due to production of a rearrangement species analogous to the tropylium ion. Cleavage between the nitrogen and methyl group alpha to the benzyl ring gives this fragment and is typical of alkyl substituted benzenoid compounds. High resolution mass spectrometry was also done due to variable results obtained with elemental analysis. These double-focusing sector mass spectrometers use a magnetic field in series with an electrostatic field to focus the ions and reduce band spreading due to differences in kinetic energy. This provides high resolution mass information (R as high as 40,000). No spectra were supplied but a table of the more prominent peaks and their abundancies are provided for both I C T B A analogs (See Table 12 and Table 13). As expected, the electron impact ionization mode resulted in several fragments. W i t h p - I C T B A , the molecular peak was obtained at m/z 371.10989 (calculated 371.11099), abundance 4.05%. This within 0.0003% of the calculated mass. The base peak was at m/z 302.03947 and likely corresponds to loss of the pentyl group. Additional significant peaks were present at m/z 356.08720 (16.36%) and m/z 245.97695 (88.89%). The former may be due to loss of the methyl group and the latter, loss of both the tertiary butyl and pentyl groups. With o-ICTBA, the molecular peak was seen at m/z 371.11060 with an abundance of 2.80%o. This is within 0.00001%) of the calculated mass. Additional peaks occurred at m/z 303.04357 (12.31%), m/z 302.03996 (90.76%) and 245.97740 (base peak). These 90 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS likely correspond to loss of a methyl groups for m/z 303.04357, loss of the pentyl for m/z 302.03996, and loss of the tertiary butyl and pentyl groups for m/z 245.97740. Table 12. Atomic Composition Report for o-ICTBA following High Resolution Mass Spectrometry Ion Mass, Observed (m/z) Abundance Fragment identity Calculated Mass Formula 245.97740 base loss o f pentyl and /-butyl 245.97798 C 8 H 9 N I 302.03996 90.76 loss o f pentyl 302.04059 C 1 2 H 1 7 N I 356.08692 16.01 loss o f methyl 356.08752 C 1 6 H 2 3 N I 303.04357 12.31 loss o f pentyl/protonated 303.04840 C 1 2 H 1 8 N I 371.11060 2.80 molecular ion 371.11099 C I 7 H 2 6 N I Note: Instrument = Kratos M S 50, Total number of peaks = 72, Temperature = 150°C, Voltage = 70 e V Table 13. Atomic Composition Report for p-ICTBA following High Resolution Mass Spectrometry Ion Mass, Observed (m/z) Abundance Fragment identity Calculated Mass Formula 302.03947 base loss o f pentyl 302.04059 C 1 2 H 1 7 N I 245.97695 88.89 loss o f pentyl and /-butyl 245.97798 C 8 H 9 N I 356.08720 , 16.36 loss o f methyl 356.08752 C 1 6 H 2 3 N I 303.04234 13.34 loss o f pentyl/protonated 303.04840 C I 2 H I 8 N I 371.10989 4.05 molecular ion 371.11099 C I 7 H 2 6 N I Note: Instrument = Kratos M S 50, Total number of peaks = 72, Temperature = 150°C, Voltage = 70 e V 2.3.2.4. Infrared Spectroscopy Organic compounds generally produce very complex infrared (IR) spectra. However, specific groups produce characteristic vibrations and this can be an application for IR if they are not well demonstrated by other spectroscopic methods. Additionally for this project, the technique was inexpensive and readily available. Initially, IR was used to confirm the presence of the amine proton of IBTAmine following borane reduction of 91 Laura N . A l c o r n V-^ [ L Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS IBTAmide. The exchangeable hydrogens attached to the nitrogen lead to unreliable chemical shifts on N M R and they usually resonate over a wide range from 5.0 to 12.0 ppm 1 2 6 . The resulting signal is also usually flattened and sometimes unidentifiable on the N M R spectrum. On the IR spectrum, the N - H stretching vibration can be seen near 3300 cm"1 although it too, can be very weak and difficult to detect. A broad, medium strong signal is usually obtained for the N - H deformation vibration at 650-900 cm"1. Unfortunately, aryl hydrogens also produce a signal in the 3030 cm"1 region as can be seen in the spectrum for o - ICTBA (Figure 31), page 116. This proved to be a problem in the IR spectrum for IBTAmine with an equivocal area in the region of the N - H stretching vibration and interference due to the other bonds in the N - H deformation vibration area. Mass spectrometry and elemental analysis was required to confirm the presence of the amine linkage. On the IR spectrum of o-ICTBA, additional signals are present in the fingerprint region between 465 cm"1 and 1600 cm"'and are likely due to aryl ring vibration (1450-1600 cm 1 ) , C - N stretch (1020-1220 cm 1 ) and C-I stretch (465-600 cm 1 ) . 2.3.2.5. Radiochemical Analysis The radiolabeling efficiencies of the exchange reaction were 90% for o - I C T B A and 98%) with p - I C T B A . Further purification of the ortho species gave a final product with less than 3% free radioiodine. This was achieved by passage through a C 1 8 Sep Pak (Glen Research Poly Pak™ cartridge). Careful stepwise elution using ethanol to initially remove free iodine and then collection of the radiolabeled product was necessary. H P L C analysis indicated that radioiodinated cuprous iodide may have been present in the final eluate as well with a retention time of between 6 and 8 minutes (See Figure 26 and Figure 92 Laura N . A l c o r n ^ IL Muscarinic Radiopharmaceutical Chapter 2. SYNTHESIS 28, pages 113 and 114). This is likely since the radioactivity recording shows at least two or perhaps three peaks in the region expected to contain free iodine. If only free iodine was present, a single peak should be present. Further analysis using H P L C could have been done to determine the identity of these contaminants but was not pursued due to 1 their low levels in the final preparation. The radiochemical purity for the ortho and para analogs were 98.8% and 98.9%), respectively. The overall radiochemical yields for each analog were 42% for o - I C T B A and 98.9% for p - I C T B A . The specific activity of the final preparations was low at a maximum of 122 GBq/mmol (3.34 Ci/mmol). Specific activity could only be approximated for the ortho analog due to the Sep Pak purification step. In this project, the theoretical maximal specific activity is about 7,400 TBq/mmol. A n approximate human dose of 370 M B q would contain 0.05 pmole of ligand available for interaction and binding with the total body muscarinic receptor population. Usually, the specific activity achievable with this labeling technique is in the range of 37 TBq/mmol, which results in a total administered dose of 10 pmole of ligand. The iododestannylation approach has provided high specific activities and was tried for this project but unfortunately could not be used due to inability to prepare the stannylated precursor. 93 Laura N . A l c o r n r^ -11L Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE Chapter 3. BIODISTRIBUTION IN MICE 3.1 Experimental 3.1.1. A n i m a l protocols Male C D - I / U B C mice weighing 25-30g received by injection into a lateral tail vein, 110 kBq of either 1 2 3 I -o -ICTBA or 1 2 3 I - p - I C T B A in 100 uL of 5 % aqueous ethanol. Each dose contained approximately 9 x 10"10 moles of ligand. At selected intervals (15 min., 1 nr., 2 hr., 4 hr., and 6 hr.) following the injections, the animals were anesthetized with halothane and quickly sacrificed by decapitation. Immediately after death, blood was collected from a neck vessel. Five mice per time point were injected. The desired tissues and whole organs were harvested (blood, liver, kidneys, small intestine, adrenal gland, stomach, lung, heart and muscle), rinsed with water, blotted dry and added to preweighed tubes. The latter were reweighed to provide tissue weights and subsequently counted in a Packard (Canberra, Canada) Cobra II Auto-Gamma counter. The tail was also harvested and counted for radioactivity which was assumed to be interstitial and therefore subtracted from the total body dose to give the net dose injected. The fractional accumulation of radioactivity in the tissue (expressed as % ID/g) was determined by comparison of tissue radioactivity with suitable dilutions of the injected dose. Tissue:blood ratios were determined from the % IDA* values of selected tissues. Finally, the fractional accumulation of radioactivity for selected organs (% ID/organ) was obtained from the product of the organ weights and % ID dose/g of tissue. For the blocking studies, two groups of mice were all preinjected with 1 mg/kg atropine, intramuscularly into a leg muscle and 30 minutes later, received 110 kBq of 94 Laura N . A l c o r n r-^ -11L Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE either , 2 3 I - o - I C T B A or l 2 3 I - p - I C T B A as above. Animals in both groups were sacrificed at the same time intervals post injection as previously used. Tissue harvesting and data analysis was also done similarly. A custom biodistribution computer program developed by Mr. Peter Taylor, Division of Nuclear Medicine, Vancouver General Hospital, was used to calculate percent injected dose for each tissue. A brief description of the program is given in Appendix 3. Biodistribution Program, page 123. 3.1.2 Statistical Methods. The two-tailed Student's T-test was used to compare data between groups and provide p-values. Best-fit curves were generated using Slide Write™ software (Advanced Graphics Inc., Carlsbad, C A ) . 3.2 Results and discussion (See Appendix 2. Summary of Biodistribution Results, page 117 for tables summarizing the results of the biodistribution experiments.) The o - I C T B A data is given in Table 17, page 117 and Table 18, page 118. These tables summarize the percent of radioactivity taken up by the selected organs per gram of tissue. Table 17 gives the data for the unblocked animals and Table 18, the atropine blocked group. Table 21 and Table 22, page 121 give the same data expressed as percent injected dose per total organ. The p - I C T B A data for percent of radioactivity taken up by the selected organs per gram of tissue is given in Table 19, page 119 and Table 20, page 120. Table 19 gives the 95 Laura N . A l c o r n V - ^ I L Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE data for the unblocked animals and Table 20, the atropine blocked group. Table 23 and Table 24, page 122 give the same data expressed as percent injected dose per total organ. As was done for the o - ICTBA data, ratios have been calculated for the difference between heart and blood; heart and lung; and adrenal and blood uptake. These are significant since they allow assessment of the target-to-nontarget contrast that may be expected on external images. Little comparative data in mice using other muscarinic radiopharmaceuticals could be found in the literature. Several reports of rat biodistribution have been published and will be compared to the results obtained with the I C T B A analogs. 3.2.1 Unblocked Studies The highest concentrations of radiotracer are observed with both the ortho- and para-analogs in the heart, liver, adrenal gland, and small intestine. This was particularly true with the para species. Uptake in the remaining organs was unremarkable and paralleled that of the circulation. Radioactivity clears rapidly in the first 1 to 2 hours. Compared to the bretylium analog RIBA, I C T B A uptake was greater in all tissues at the same time points post-injection. 3.2.1.1. Heart Localization. R I B A data for myocardial accumulation in animals is available for the rat, pig and dog. In the rat, heart uptake has been reported to be as high as 0.208 ± .014 %ID/g at 2 hours post-injection, compared to 0.24 ± .08 %ID/g with o - ICTBA and 0.49 ± .14 %ID/g with p - I C T B A . O f the two available R I B A studies in the rat, marked variability in biodistribution results between these reports was found 1 , 1 0 0 . Tissue uptake differed by a 96 Laura N . Alco rn i—11 Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE factor of ten for almost all organs between these two studies. Blood uptake was significantly less than the highest value obtained for R I B A (0.005 ± <.001 %ID/g) compared to 0.34 ± 0.06 %ID/g with o - ICTBA and 0.39 ± .07 %ID/g with p - I C T B A at 2 hours post-injection. Figure 16 gives the time course of heart and blood radioactivity with o-ICTBA. Figure 17 is the comparable graph obtained with p - I C T B A . For both analogs there was greater heart than blood uptake only at 15 minutes post-injection. At all other time points, the relationship was less than one. Both curves are biphasic with an initial fast distribution phase followed by a slower phase. The transition to the slow phase occurs earlier with o - ICTBA at about 1 hour post-injection compared to 2 hours with p - I C T B A . Ortho, Unblocked, Heart vs Blood 2.00 1.60 h E heart blood 0.00 0 2 3 4 5 6 7 Time (hrs) Figure 16. o-ICTBA, Percent Injected Dose/gram Vs Time Post-injection (hr.): Heart Vs Blood Although higher heart uptake was obtained with I C T B A compared to R I B A , the heart to blood ratio at 15 minutes is much lower at 1.63 (o-ICTBA) and 1.54 ( p - I C T B A ) 97 Laura N . A l c o r n <—11 Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE compared to R I B A at 41.6. Similarly, the heart to lung ratios are lower in this study at 1.29 for ortho and 0.86 for para compared to 6.5 with RIBA. Minimally, a ratio of approximately 5 is required for sufficient contrast in a nuclear medicine image of the heart67. Persisting blood levels of I C T B A may be due to slow metabolic clearance or Para, Unblocked, Heart vs Blood 3 T CO 2 O) "5 <A \ i A heart TD TJ CD 7j T\ • blood * 1 i i i i i i 0 1 2 3 4 5 6 7 Time (hrs) Figure I7. p-ICTBA, Percent Injected Dose/gram Vs Time Post-injection (hr.): Heart Vs Blood delayed accumulation in the target tissues. This is also a problem with the Q N B analogs. In patients with Alzheimer's disease, imaging cannot be performed until 21 hours post-injection of radioiodinated Q N B 5 6 ' 9 2 . The N-methyl analog, M Q N B has been investigated as a heart imaging agent using PET. The tritiated analog would be expected to behave much more like the parent compound due to the identical physico-chemical properties of the two analogs. Use of the radioiodine label might interfere with the interaction between the receptor and ligand, potentially decreasing uptake in target organs. Comparative 98 Laura N . A l c o r n c=LIL Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE uptake in the heart, lungs and blood are given for two stereoisomers of Q N B as well as tritiated M Q N B in the rat at 15 minutes, 2 and 4 hours post-injection in Table 14. Table 14. Biodistribution of QNB and ICTBA analogs Percent Injected dose per Gram Tissue Time Post-Injection 15 minutes 2 hours 4 hours Ref. Ana log Heart Lung Blood Heart Lung Blood Heart - Lung Blood -84 R , R -Q N B 1.34 5.66 0.075 0.320 1.130 0.051 0.120 0.672 0.126 98 R ,S -Q N B iisis 2.69 0.066 0.151 1.62 0.085 0.080 0.608 0.116 70 M O N B * 2.35 0.184 0.075 0.60 0.103 0.072 0.46 0.14 0.075 o - I C T B A 1.34 1.04 0.825 0.244 0.382 0.336 0.208 0.382 0 274 p - I C T B A 2.29 2.66 1.48 0.489 0.963 0.390 0.432 0.855 0.353 t tritiated Heart uptake with both I C T B A analogs is comparable to the other Q N B analogs at each time point but blood levels remain much higher, resulting in poor heartblood ratios (see Table). Lung uptake of both I C T B A species is lower than the Q N B analogs which gives larger target-to-nontarget organ ratios especially for ortho-ICTBA. As expected, the " tritiated M Q N B data demonstrate very high target organ uptake and target-to-nontarget ratios70. However, the ratios for both I C T B A species remain less than one at all time points except the 15 minute result for o-ICTBA. These results indicate that neither the ortho or para analog would provide adequate heart to background ratios to allow assessment of receptor distribution by external imaging. 99 Laura N . A l c o r n c=U Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE Table 15. Target-to-Nontarget Ratios with QNB and ICTBA analogs Target-to-Nontarget Ratios Time Post-Injection 15 minutes 2 hours 4 hours Ref. Ana log Heart:Blood Heart: Lung Heart:Blood Heart: Lung Heart:Blood Heart: Lung 84 R , R -Q N B 0.24 6.27 0.28 0.95 0.18 98 R ,S -Q N B 11.2 0.28 1.78 0.09 0.69 0.13 70 M Q N B 1 31.3 12.8 8.3 5.8 6.1 3.3 o - I C T B A 1.63 1.29 0.73 0.64 0.76 0.52 p - I C T B A 1.54 0.86 1.25 0.51 1.23 0.51 t tritiated. 3.2.1.2. Liver Localization. Liver uptake is high for the I C T B A analogs at the 15 minute point and decreases with time after this point (see Table 17, page 117 and Table 19, page 119). Uptake with o - ICTBA was 3.41 ± 0.97 %ID/g at 15 minutes, 1.54 ± 0.25 %ID/g at 1 hour, 1.09 ± 0.35 %ID/g at 2 hours, 1.05 ± 0.27 %ID/g at 4 hours, and 0.53 ± 0.34 %ID/g at 6 hours. With the para analog it was 13.01 ± 0.95 %ID/g at 15 minutes, 9.17 ± 1.53 %ID/g at 1 hour, and 6.69 ± 0.74 %ID/g at 2 hours, 7.64 ± 0.77 %ID/g at 4 hours, 6.74 ± 0.97 %ID/g at 6 hours. The observed accumulation in the liver is likely related to the metabolic pathway for the two species. 3.2.1.3. Adrenal Localization. The results from both o - ICTBA and p - I C T B A indicate the adrenal gland concentrated and retained the radioactivity to the greatest extent. The adrenakblood ratio peaked 4 hours post-injection at 8.3 with the ortho and 14.5 with the para (see Table 16). 100 Laura N . Alco rn ^ IL Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE At all time points post-injection, adrenal uptake remained significantly higher than blood (p<0.05). The adrenal and blood radioactivity curves for the ortho and para analogs are given in Figure 18 and Figure 19, respectively Adrenal localization was also seen in the preliminary research using o -BIBTA and in work done with RIBA.. With o-BIBTA, adrenal uptake peaked at 60 minutes at 61.1 ± 16.2 % ID/g, which is much higher than the results for o - ICTBA (3.28 ± 1.24 % ID/g) and p - I C T B A (15.94 ± 7.72 % ID/g). As observed in the heart, adrenal uptake with the I C T B A analogs is much greater than that reported for RIBA: 0.364 ± 0.048 % ID/g at 6 hours post-injection for R I B A compared to 1.47 ± .57 for o - ICTBA and 6.47 ± 1.77 forp-I C T B A . However, because of higher blood levels with o - ICTBA (see Table 17, page 117) the adrenahblood ratio is less at 4.61 versus 7.1 with RIBA. This is not the case for p - I C T B A with an adrenakblood ratio of 10.2 at 6 hours post-injection (Table 19, page 119). The radioiodinated R,S diastereomer of Q N B was taken up in the adrenal gland to a moderate degree in rats. Uptake was highest 15 minutes post-injection at 1.98 % ID/gram and decreased rapidly at all time points measured up to 24 hours post-injection. The adrenakblood ratios were very high due to low levels in the circulation. Table 16 gives the adrenakblood ratios at 15 minutes, 1 hour, 2 hours, 4 hours and 6 hours if available for some Q N B , RIBA and I C T B A analogs. With the exception of the first ratio, all values for the Q N B analog are less than those obtained with o - I C T B A and p - I C T B A . The results for p - I C T B A are much higher than those for o -ICTBA. For both I C T B A analogs the ratio increases after 15 minutes to a peak at 4 hours, unlike the Q N B 101 Laura N . A l c o r n .—11 Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE analog. Although the target-to-nontarget ratios are high, the total organ uptake is very low for both I C T B A analogs (see Table 21 and Table 23, pages 121 and 122). With only Table 16. Adrenal:Blood Ratios with QNB and ICTBA analogs AdrenahBlood Ratio Time Post-Injection Ref. Ana log 15 minutes 1 hour 2 hours 4 hours 6 hours . 98 R , S - Q N B 30 5.06 2.97 1.71 1.54 100 R I B A n/a n/a n/a n/a 7.1 124 o - B I B T A n/a 8.48 17.7 n/a n/a o - I C T B A 4.35 7.34 5.78 8.3 4.61 p - I C T B A 12.0 11.6 8.00 14.5 10.2 n/a = not available 0.05 to 0.16 percent injected dose per total organ, external imaging would not be possible. Another concern with interpretation of the adrenal results is the high variability in the data This may be due to the difficulty of dissecting and weighing such a small organ. In the mouse, the two glands together typically weigh between 5 and 25 mg. The adrenal gland is thought to contain m l and m2 subtypes of the muscarinic receptor (see page 13 in Section 1.1.2, Distribution). However, the blocking experiment did not demonstrate any attenuation of adrenal uptake after pre-injection of atropine. This would suggest that the observed localization was not due to muscarinic receptor interaction. Possibly some other receptor system or marker found in high concentrations in the adrenal gland was bound by the radiopharmaceutical. Additional blocking experiments with other non-specific antagonists could be done to determine which receptor system was involved. 102 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE Ortho, Unblocked, Adrenal vs Blood A adrenal • blood 0 1 2 3 4 5 6 7 Time (hrs) Figure 18. o-ICTBA, Percent Injected Dose/gram Vs Time Post-injection (nr.): Adrenal Vs Blood Para, Unblocked, Adrenal vs Blood \ JU 24 lose/gram 18 -I '—^ A blood TD B © 12 • adrenal 6 i I i —A— ] A 1 A 0 2 3 4 5 6 Time (hrs) 7 Figure 19. P-ICTBA, Percent Injected Dose/gram Vs Time Post-injection (nr.): Adrenal Vs Blood 103 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE 3.2.1.4. Small Intestine Localization. Radioactivity increases with time in the small intestine with the ortho analog, possibly reflecting the metabolic fate of the radiopharmaceutical. This is not evident however, with the para analog (see Table 17, page 117 and Table 19, page 119). Uptake with o - I C T B A was 2.92 ± 0.46 %ID/g at 15 minutes, 1.41 ± 0.48 %ID/g at 1 hour, 1.14 ± 0.47 %ID/g at 2 hours, 1.23 ± 0.29 %ID/g at 4 hours, and 0.87 ± 0.38 %ID/g at 6 hours. With the para analog it was 3.11 ± 0.76 %ID/g at 15 minutes, 3.02 ± 0 . 7 % I D / g at 1 hour, 2.06 ± 0.32 %ID/g at 2 hours, 2.02 ± 0.34 %ID/g at 4 hours, and 1.51 ± 0.43 %ID/g at 6 hours. The small intestine is considered to be primarily a site for the m3 and to a less extent, the m2 subtypes. 3.2.2 Ortho versus Para Distr ibution A n initial observation in comparing the ortho and para analogs is the marked difference in tissue uptake. P - I C T B A uptake was consistently much higher than o-I C T B A . The only difference in the preparation of the two radiopharmaceuticals was the purification step applied in the preparation of the ortho analog. Due to a lower labeling efficiency the ortho species was passed through a C-18 styrene Sep-Pak after incubation with the radioiodine to remove persisting free iodine. In the adrenal gland, work with R I B A has shown that moving the iodine atom from the ortho to the para position markedly decreased localization in two studies 1 1 0 0 and resulted in improved adrenal localization in another". With the I C T B A analogs, adrenal 104 Laura N . A l c o r n r^ -11 Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE uptake with the ortho analog was three to five times less than the para (see Table 17, page 117 and Table 19, page 119). Additionally, the adrenakblood ratios were consistently lower with the ortho analog (see Table 16). In the heart, the effect of position of the iodine on the phenyl ring of R I B A is thought to be one favoring ortho rather than para\ Again, para uptake was consistently higher than ortho in the heart with the I C T B A analogs (see Table 14). Brain uptake has been found to be influenced by iodine position on the aromatic ring in a series of amines studied in the rat69. The highest organ localization values and brain-to-blood ratios were obtained for the para, and lowest for the ortho. This was also true at all time points with the I C T B A analogs. A l l five muscarinic receptor subtypes have been found in the brain. Interestingly, brain uptake of R I B A was consistently less than 0.001 % ID/g for the ortho, meta and para analogs at all time points, post-injection. Low brain uptake with R I B A may be related to poor penetration of the blood:brain barrier with this quaternary amine. 3.2.3 Effect of Atropine Blockade With the possible exception of the para analog in the heart and brain, atropine blockade resulted in either no significant change in organ uptake or in one case, increased uptake. The effect of atropine blockade on heart uptake of p - I C T B A is shown in Figure 20. At all time points except the 2 hour, uptake in the heart is significantly attenuated by atropine blockade (p<0.05). The degree of attenuation was 42% at 15 minutes, 56% at 1 hour, 0% at 2 hours, 37%) at 4 hours and 43% at 6 hours. Attenuation is most evident at the 15 minutes and 1 hour time points. Figure 21 shows the effect of atropine blockade 105 Laura N. Alcorn c=U Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE on brain uptake of p - I C T B A . Again, except for the 2 hour time point, organ uptake was significantly less after atropine administration (p<0.05). The attenuation was Para, Heart, Unblocked vs Blocked \ 3 T K 1 \j - K K x — "— A A I I l I ) 1 2 3 4 5 6 7 Time (hrs) A unblocked • blocked Figure 20. P-ICTBA, Percent Injected Dose/gram vs Time Post-injection (hr.) in the Heart: Blocked vs Unblocked approximately 35% for this organ except for 0% at 2 hours. Muscarinic ligands with very slow dissociation rates may not be competitively displaced by atropine, particularly if binding determinations are made too early after administration of the competing ligand 1 2 7. For example, in rat heart muscarinic receptors, organ uptake was significantly less after atropine administration (p<0.05). The R-[ 3 H]QNB has a dissociation rate constant of 77 minutes and is not displaced by atropine even up to 4 hours after addition of this competitive ligand. 106 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE Para, Brain, Unblocked vs Blocked unblocked blocked 0 1 2 3 4 5 6 7 Time (hrs) Figure 21. P-ICTBA, Percent Injected Dose/gram vs Time Post-injection (hr.) in the Heart: Blocked vs Unblocked Ortho, Lung, Unblocked vs Blocked 2.80 E m o> 0 2.10 0.70 0.00 -A unblocked 1 • blocked "T K ———i— i 3 4 Time (hrs) Figure 22. O- ICTBA, Percent Injected Dose/gram Vs Time Post-injection (hr.) in the Lung: Blocked vs Unblocked A n unexpected result was the increased uptake of the ortho analog in the lung after atropine blockade (Figure 22). This tissue is considered a site of predominantly m3 107 Laura N. Alcorn i—11 Muscarinic Radiopharmaceutical Chapter 3. BIODISTRIBUTION IN MICE and m4 muscarinic subtypes. It is unclear why lung uptake increased in response to blockade of muscarinic receptors. It is possible that the ortho analog binds to some other non-muscarinic receptor and displacement by atropine of nonspecific binding at the muscarinic receptor resulted in increased uptake at the unknown receptor. 108 Laura N . A l c o r n Muscarinic Radiopharmaceutical Chapter 4. SUMMARY Chapter 4. SUMMARY 1. Reliable methods for the synthesis of o -ICTBA and p - I C T B A were devised. The overall yield for o - ICTBA was 39% and for p - I C T B A was 37%. 2. Identity testing for ortho and para I C T B A consisted of thin layer chromatography, nuclear magnetic resonance spectrometry, high performance liquid chromatography and elemental analysis. (9-ICTBA underwent additional analysis with mass spectrometry and infrared spectroscopy. A l l analytical procedures confirmed the identity of o - ICTBA and p - I C T B A . 3. Radiolabeling with 1 2 3I was accomplished using the Cu(I) exchange labeling method. The radiochemical purity for the ortho and para analogs was 98.8%) and 98.9%>, respectively. The radiochemical yield for o - ICTBA was 42% and for p - I C T B A was 98.9%). The specific activity of the final preparations was low at a maximum of 122 GBq/mmol (3.34 Ci/mmol). The theoretical maximum is 7,400 TBq/mmol. At least 37 TBq/mmol is desirable for ex-vivo imaging. 4. Biodistribution experiments were conducted with both radioiodinated analogs in mice. Ten tissues were assessed at 0.25, 1, 2, 4 and 6 hours post-injection. The following organs were harvested: blood, liver, kidneys, small intestine, adrenal gland, stomach, lung, heart, and muscle. Curves were generated of percent injected dose per gram of tissue versus time post-injection. 109 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapter 4. SUMMARY 5. Comparison was made between target organs and background tissues. The heart to blood ratio for the ortho derivative was a maximum of 1.63 and for the para, was 1.5 at 15 minutes post-injection. Uptake rapidly declined however, and the ratios fell to below one for all subsequent sampling times. The target-to-nontarget ratio usually must be at least 5:1 to permit external imaging of the heart. With both o - ICTBA and p - I C T B A the adrenal gland concentrated and retained the radioactivity to the greatest extent. The adrenakblood ratio peaked at 4 hours post-injection at 8.3 with the ortho and 14.5 with the para. 6. Distribution of the ortho and para analogs was also compared and found to be distinctly different. Organ uptake was consistently much higher with p - I C T B A . The only difference in the preparation of the two analogs was a purification step used in the radiolabeling procedure for the ortho analog. 7. A series of mice received either o - ICTBA or p - I C T B A following administration of atropine, a muscarinic receptor blocking agent. Comparison of target tissue uptake was made with and without receptor blockade. Atropine pre-injection was not associated with attenuation of radioligand accumulation in receptor-bearing tissues after administration of the ortho analog. In the animals given the para analog, a decrease in radioligand uptake was observed in the heart (approximately 40%) and brain (35%>) at all sampling times except the two hour. Synthesis and radioiodination of o - ICTBA and p - I C T B A were performed in order to assess these two radiopharmaceuticals as potential heart imaging agents. The synthesis was reliable and proceeded with acceptable yields. The specific activity obtained 110 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Chapters SUMMARY following radioiodination was less than ideal. Biodistribution studies in mice indicated with the para species that uptake in the heart, brain and adrenal gland may be due to muscarinic receptor binding. Insufficient target to nontarget ratios were obtained with both analogs to allow external imaging. Further investigation of p - I C T B A including determination of it's receptor binding kinetics could be done to identify the particular cell constituent responsible for the observed heart, brain and adrenal localization. Subsequently, structural modifications to the molecule could be tried to improve target organ localization. Use of other radioiodination techniques may increase the specific activity. Ill Laura N . A l c o r n Muscarinic Radiopharmaceutical Appendix 1. Spectral Analysis. Appendix 1. Spectral Analysis. OStHD/PtNtflfl JNE/CDCLl JLJLA Figure 23. 200 MHz Proton magnetic resonance spectrum of ortho-ICTBA in CDCI3 PARAPeWIflMJNt/CDCU r. 1 • n • [ M ! 1 1 I1; rt M " " ! " " ! " " ! ' 'I ' ''''' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' fl 0 ;.S ?. 8 6.S 6.0 5.5 S. 8 1.5 1.0 3.5 3. 8 2.5 3.A 1.5 1.8 .5 8.0 Figure 24. 200 MHz Proton magnetic resonance spectrum of para-ICTBA in CDCI3 112 Laura N . A l c o r n Muscarinic Radiopharmaceutical Appendix 1. Spectral Analysis. V 3 | 3^ ^» ™> CVJ LT9 (_K> Figure 25. UV Recording of Reverse phase High Performance Liquid Chromatograph of ortho-ICTBA . Chromatographic conditions: column: Ultremex Semi-prep column (250x10mm), mobile phase: 95% Ethanol 5% 2MTEA; HPLC flow rate: 2.0 ml/min.; ultraviolet detection wavelength: 254 nm. • J — T Z — ~ — — - + , , ..„. *» . .O • . . . . 0 LS" « -> o. tx ' O. * K rO K n r~ ' J - - -• % - " 1 c < ™ •< t-i. o. .P< ' -O. .O > 2 CO * . 00 - > • co cu — < „ - ^ < V - 4 " --<' .—. - -• -» -ft • I* lb 14 to S • » Hll Li Figure 26. Radioactivity Recording of Reverse phase High Performance Liquid Chromatograph of ortho-ICTBA . Chromatographic conditions: column: Ultremex Semi-prep column (250x10mm), mobile phase: 95% Ethanol 5% 2M TEA; HPLC flow rate: 2.0 ml/min.; Chart rate: lem/min. 113 Laura N . A l c o r n Muscarinic Radiopharmaceutical Appendix 1. Spectral Analysis. Figure 27. UV Recording of Reverse phase High Performance Liquid Chromatograph ofpara-ICTBA . Chromatographic conditions: column: Ultremex Semi-prep column (250x10mm), mobile phase: 95% Ethanol 5% 2MTEA; HPLC flow rate: 2.0 ml/min.; ultraviolet detection wavelength: 254 nm. Figure 28. Radioactivity Recording of Reverse phase High Performance Liquid Chromatograph ofpara-ICTBA. Chromatographic conditions: column: Ultremex Semi-prep column (250x10mm), mobile phase: 95% Ethanol 5% 2MTEA; HPLC flow rate: 2.0 ml/min.; Chart rate: lcm/min. 114 Laura N . A l c o r n Muscarinic Radiopharmaceutical Appendix 1. Spectral Analysis. 372.15 100 % 37S.H 115.94 1 0 160 180 ' 200 220 240 ' 260 ' 280 ' 300 320 340 360 3B0 400 420 440 Figure 29. Atmospheric Pressure Chemical Ionization Mass Spectrum of ortho-ICTBA . Scan conditions: Source temperature: 100° C; Cone voltage: 25 V; Sample: 20 ppm in methanol (10 pLofa solution of 2 mg/ml in CH2CI2 in 1 ml MeOH); Scanning speed: 100 amu/sec 100 : 100 3U.21 37I.M 21693 200 450 Figure 30. Positive electrospray Mass Spectrum of ortho-ICTBA . Scan conditions: Source temperature: 100° C; Cone voltage: 30 V; Sample: 10 ppm in methanol and aq. Formic acid (10 pLofa solution of 2 mg/ml in CH2CI2 in 1 ml MeOH, added in equal parts to a mixture of 50% water + 0.1% formic acid); Scanning speed: 100 amu/sec; Note: the peaks at m/z 316.21 and 371.93 are off-scale. 115 Laura N . A l c o r n Muscarinic Radiopharmaceutical Appendix 1. Spectral Analysis. Laura N. Alcorn i—'1 Muscarinic Radiopharmaceutical Appendix 2. Summary of Biodistribution Results Appendix 2. Summary of Biodistribution Results Each time point is n-5 unless otherwise indicated. 1. Percent Injected Dose per Gram 1.2 Ortho-ICTBA Table 17. Murine Biodistribution of Ortho-l^^I CTBA (cyclopentyltertiarybutylbenzylamine) % injected dose per gram tissue ± 1 standard deviation time post-injection Tissue 15 min 1 hr 2hr 4hr 6hr Blood 0.82 ±0.13 0.45 ±0.05 0.34 ±0.06 0.27 ±0.02 0.32 + 0,29 Liver 3.41 ±0.97 1.54 ±0.25 1.09 ±0.35 1.05 ±0.27 0 53 ±0.34 Kidney 0.98 ± 1.25 1.20 ±0.31 0.80 ±0.25 0.78 ±0.18 0.66 ±0.19, Small Intestine 2.92 ± 0 46 1.41 ±0.48 1.14 ±0.47 1.23 ±0.29 0.87 ±0.38 Adrenal 3 59 ± 2 37 3.28 ± 1.24 1.94 ± 1.02 2.27 ± 1.25 1 47 ± 0.57f Stomach 0 87 ±0.30 1.09 ±0.34 1.04 ±0.41 1.09 ± 0.99 0.42 ±0.26 Lung 1 04 ± 0.26 0.55 ±0.08 0.38 ±0.08 0.40 ±0.08 0 20 ±0.09 Heart 1 34 ±0.55 • 0.41 ±0.09 0.24 ±0.08 0.21 ±0.05 0.11 ±0.07 Muscle 0 50 ±0.18 0.35 + 0.05 0.23 ±0.12 0.28 + 0.12 0 22 ±0.16 Brain 0.35 ±0.18* 0.24 ±0.04 0 20 ±0.06 0.17 ±0.02 0 13 + 0.07 Heartblood 1.63 0.92 0 73 0.76 0 35 Heart: lung 1 29 0.74 0.64 0.52 0 55 Adrenal:blood 4.35 • '7.34 5.78 8.30 4.61 fn=4 117 Laura N . A l c o r n Muscarinic Radiopharmaceutical Appendix 2. Summary of Biodistribution Results Table 18. Murine Biodistribution of Ortho-^^I CTBA (cyclopentyltertiarybutylbenzylamine) Post Atropine Blockade % injected dose per gram tissue ± 1 standard deviation time post-injection Tissue 15 min 1 hr 2 h r 4 h r 6hr Blood 0.57 'M 0.16 0.19 + 0 03 0.14 ±0.04 0.17 ± 0.03 0.15 ll 0.05 Liver 6.51 ISr 1.45 1.69 ± 0 29 1.36 + 0.49 1.52 ± 0.12 1.14 n 0.29 Kidney 3.67 Iffi 1.06 1.30 ± 0 30 0.78 ±0.20 0.91 ± 0.17 0.70 0.20 Small Intestine 1.82 0.28 2.07 ± 0 99 2.04 ±0.85 1.43 ± 0.40 1.00 ;± 0.41 Adrenal 4.14 + 1.10 2.22 ± 1 08 2.36 ±0.97 3.43 ± 0.93 1.95 Ii 0.94 Stomach 0.75 IS 0.30 0.59 ± 0 24 0.41 ±0.13 0.43 ± 0.04 0.36 0.07 L u n g 1.13 i§ 0.26 0.49 ± 0 31 0.37 ±0.12 0.37 ± 0.08 0.27 0.08 Heart 1.52 "M 0.42 0.33 ± 0 07 0.19 ±0.06 0.21 ± 0.05 0.11 si 0.04 Muscle 0.80 iij 0.19 0.45 ± 0 24 0.31 ±0.16 0.41 ± 0.11 0.33 ft 0.15 Brain 0.54 •s 0.08 0.29 ± 0 11 0.26 ±0.11 0.21 ± 0.02 0.11 is 0.02 Heart:blood 2.68 1.76 1.36 1.29 0.76 Heart: lung 1.34 0.68 0.52 0.58 0.42 Adrenahblood 7 31 11.74 16.6 20.7 13.1 118 Laura N . A lco rn ' i—11 Muscarinic Radiopharmaceutical Appendix 2. Summary of Biodistribution Results 1.2 Para-ICTBA Table 19. Murine Biodistribution of Para-^^ICTBA (cyclopentyltertiarybutylbenzylamine) % injected dose per gram tissue ± 1 standard deviation time post-injection Tissue 15 min 1 hr 2 h r 4 h r 6 h r B lood 1.48 ±0.09 - 1.37 ± 0.37 0.39' ±0.07 0.35 ± 0.07 0.63 ± 0.11 Liver 13.01 ±0.95 9.17 ± 1.53 6.69 ±0.74 7.64 ± 0.77 6.74 ± 0.97 Kidney 6.70 ±0.52 4.61 ± 1.33 3.18 ±0.67 2.83 + 0.54 2.56 ± 0.43 Small Intestine 3.11 ±0.76 3.02 ± 0.75 2.06 ±0.32 2.02 + 0.34 1.51 0.43 Adrenal 17.87 ± 1.70 15.94 t7.72 3.12 ± 1.588t 5.11 ± 2.32 6.47 ± 1.77 Stomach 2.13 ±0.50 1.71 ± 0.78 0.78 ±0.23 0.61 ± 0.11 0.87 !:§ 0.24 Lung 2.66 ±0.30 2.27 ± 0.45 0.96 ±0.17 0.86 ± 0.13 1.21 + 0.21 Heart 2.29 ± 0.26 ' 1.33 ± 0.36 0 49 ±0.14 0.43 + 0.08 0 44 0.11 Muscle 1.73 ± 0.57 1.26 ± 0.25 0.53 ±0.14 0.80 + 0.47 0.87 l ± 0.33 Brain 1 86 ±0.11 1.10 ± 0.36 0.38 ±0.06 0.30 ± 0.03 0.31 f ± 0.12 Hear tb lood 1 54 0.97 1.25 1.23 .069 Heart: lung 0.86 0.58 0.51 0.51 0.36 Adrenal:blood 12.0 11.6 8.00 14.5 10.2 t n = 4 119 Laura N. Alcorn Muscarinic Radiopharmaceutical Appendix 2. Summary of Biodistribution Results Table 20. Murine Biodistribution ofPara-^^ICTBA (cyclopentyltertiarybutylbenzylamine) Post-Atropine Blockade % injected dose per gram tissue ± 1 standard deviation time post-injection Tissue 15 min 1 hr 2hr 4hr 6hr Blood 1.68 ±0.45 0.79 ± 0.13 , 0.68 K £ 0.16 0.40 + 0.11 0.35 0.06 Liver 6.37 ±2.16 4.74 ± 1.21 6.66 ± 1.94 4.11 ± 1.25 4.14 ± 1.01 Kidney 4.82 ± 1.37 2.68 + 0.67 2.53 n 0.58 1.57 ± 0.51 1.25 ± 0.33 Small Intestine 2.51 ± 1.16 2.46 ± 1.04 2.24 :±" 0.75 1.20 + 0.44 1 00 n 0.31 • Adrenal 14.64 ±5.85 7.05 ± 2.89 7.61 fig 1.50 4.87 ± 1.86 5.66 •I 1.88 Stomach 2.87 ±0.83 1.44 ± 0.33 1.20 w 0.09" 0.67 ± 0.16 0.40 ii 0.10 Lung 2 11 ±0.52 1.03 + 0.20 1.00 0.22 • 0.52 + 0.14 0.47 0.12 Heart 1.34 ±0.38 0.58 ± 0.12 0.50 iS 0.12 . ,0.27 + 0.07 0 25 s 0.05 . Muscle 1.76 ± 0 71 0.98 ± 0.40 0.74 0.18 0.77 + 0.39 0.62 # 0.16 Brain 1.24 ±0.38 0.60 ± 0.10 0.43 0.09 0.21 + 0.06 0.21 0.07 Heartblood 0.80 0.74 0.74 0.69 0.71 Heart: lung 0.64 : 0.57 0.50 0.52 0.53 Adrenal:blood 8.70 8.94 11.2 ' 12.3 16.0 120 Laura N. Alcorn r=LI- Muscarinic Radiopharmaceutical Appendix 2. Summary of Biodistribution Results 2. Percent Injected Dose per Organ 2.1 Ortho-ICTBA Table 21. Murine Biodistribution of Ortho-^3] CTBA (cyclopentyltertiarybutylbenzylamine) % injected dose per organ ± 1 standard deviation time post-injection Tissue 15 min 1 hr 2hr 4hr 6hr Blood 1.64 + 0.35 0.82 ±0.07 0.66 ±0.10 0.52 ±0.04 0.61 ±0.54 Liver 3.52 ± 1.28 1.42 ±0.17 1.28 ±0.33 1.25 ±0.25 1.04 ±0.42 Kidney 1.04 ±0.37 0.42 ±0.05 0.31' ±0.05 0.28 ±0.05 0.16 ±0.06 Small Intestine 0.75 ±0.53 1.10 ±0.40 0.90. ±0.43 0.83 + 0.28 0.78 ±0.39 Adrenal 0.05 ±0.03 0.03 ±0.01 0.03 ±0.01 0.04 ±0.01 0.02 ± 0.002T Stomach 0.17 ±0.05 -0.21 ±0.07 0 21 ±0.08 0.17 ±0.14 0.08 ± 0.04 • Lung 0 28 ±0.07 0.12 ±0.04 0.06 ±0.01 0.07 ±0.02 0.04. ±0.01 Heart 0.15 ±0.06 0.04 ±0.01 0.03 ±0.01 0.02 ±0.00 0.01 ±0.01 Muscle 5.71 + 2.12 3.72 ±0.45 2.60 ± 1.38 2.99 ± 1.28 2.44 ± 1.79 Brain 0.14 ±0.064* 0.12 ±0.03 0.10 ±0.03 0.09 ±0.01 0.06 ±0.03 tn=4 Table 22. Murine Biodistribution of Ortho-1231 CTBA (cyclopentyltertiarybutylbenzylamine) Post Atropine Blockade % injected dose per organ ± 1 standard deviation time post-injection Tissue 15 min 1 hr 2hr - ' 4hr 6hr Blood 1.36 ±0 .33 , 0.43 ± 0.06 0.33 ± 0.09 0.40 ± 0.08 0.38 ±0.11 Liver 13.07 ±2.50 3.07 ± 0.46 2.57 ± 0.94 2.90 ± 0.24 2.23 ±0.54'" Kidney 1.69 ±0.46 0.61 ±0.18 0.34 ± 0.09 0.41 ±0.08 0.34 ±0.11 Small Intestine 1.76 ± 0.61 2.03 ± 0.90 1.93 ±0.67 1.51 ±0.69 1.05 ±0.57 Adrenal 0.09 ± 0.02 0.04 ± 0.02 0.05 ± C 02 0.05 ± 0.02 0.03 ±0.01 Stomach 0.21 ±0.10 0.13 ±0.05 0.09 ± 0.03 0.10 ±0.02 0.09 ± 0.03 Lung 0.25 ± 0.06 0.11 ±0.07 0.07 ± 0.02 0.08 ±0.01 0.06 ±0.02 Heart 0.22 ± 0.06 0.05 ±0.01 0 03 ± 0.01 0.03 ±0.01 0.02 ±0.01 Muscle 11.14 ±2.49 5.88 ±2.96 4 . 1 5 ± 2 0 5 5.82 ± 1.61 4.85 ±2.08' Brain 0.25 ± 0.02 0.13 ±0.04 0.13 ±0.06 0.10 ±0.01 0.06 ± 0.02 121 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Appendix 2. Summary of Biodistribution Results 2.2 Para-ICTBA Table 23. Murine Biodistribution of Para-^^I CTBA (cyclopentyltertiarybutylbenzylamine) % injected dose per organ ± 1 standard deviation time post-injection Tissue 15 min 1 hr 2 h r 4 h r 6 h r B lood 3.53 ± 0 . 2 8 2.70 ± 0 . 7 4 1.03 ± 0 . 1 5 0.84 ± 0 . 1 3 1.20 ± 0 . 2 3 L ive r 20 .40 ± 2 . 1 5 11.03 ± 1.55 11.86 ± 1.30 12.20 ± 0 . 8 6 7.38 ± 1 . 7 1 Kidney 3.24 ± 0 . 4 8 1.33 ± 0 . 2 8 1.63 + 0 . 4 9 1.35 ± 0 . 2 5 0.69 ± 0 . 1 2 Small Intestine 2.59 ± 0 . 3 5 2.41 ± 0 . 6 9 2.06 ± 0 . 4 8 1.92 ± 0 . 5 9 1.18 ± 0 . 4 2 Adrenal 0.16 ± 0 . 0 9 0.16 ± 0 . 0 5 0.05 ± 0 .055 + 0.08 ± 0 . 0 6 0.08 ± 0 . 0 5 Stomach 0.48 ± 0 . 0 8 0.36 ± 0 . 1 9 0.16 ± 0 . 0 4 0.13 + 0 . 0 4 0.16 ± 0 . 0 6 L u n g 0.73 ± 0 . 1 4 0.41 ± 0 . 0 8 0.22 ± 0 . 0 3 0.23 ± 0 . 0 1 0.21 ± 0 . 0 4 Heart 0.32 ± 0 . 0 8 0.15 ± 0 . 0 4 0.08 ± 0 . 0 3 0.06 ± 0 . 0 1 0.04 ± 0 . 0 1 Muscle 23 .54 ± 6 . 8 6 14.48 ± 3 . 3 0 8.10 ± 1.60 11.12 ± 6 . 2 9 9.56 ± 3 . 6 8 Brain 0.89 ± 0 . 1 3 0.50 +0 .16 0.19 ± 0 . 0 5 0.14 ± 0 . 0 1 0.15 ± 0 . 0 6 t n = 4 Table 24. Murine Biodistribution of Para-1CTBA (cyclopentylurtiarybutylbenzylamine) Post Atropine Blockade % injected dose per organ ± 1 standard deviation time post-injection Tissue 15 min 1 hr 2 h r 4 h r 6 h r B lood 3.68 ± 1.04 1.74 ± 0.21 1.43 f 0.26 0.86 ± 0.24 0.78 ± 0 13 Liver 9.84 ± 3 31 8.12 ± 1.85 9.62 f 1.73 6.21 ± 1.62 6.11 : f 1.18 Kidney 1.94 ± 0 . 6 5 1.08 ± 0.22 0.94 f 0.22 0.59 ± 0.17 0.46 ± 0.09 Small Intestine 1.79 ± 0 . 9 5 2.45 + 1.24 2.04 f 0.78 1.12 ± 0.50 0.97 : f 0 .30 Adrenal 0.17 ± 0 . 0 7 0.08 + 0.04 0.09 ± 0.03 0.07 ± 0.03 0.07 ± 0.03 Stomach 0.55 ± 0 . 1 4 0.32 + 0.08 0.24 , : f 0.03 0.13 + 0.03 0.08 ± 0.01 Lung 0.43 ± 0 08 0.20 ± 0.03 0.19 i f 0 04 0.11 ± 0.03 0.11 i f 0 0 3 Heart 0.16 ± 0 . 0 4 0.07 + 0.01 0.06 i f 0.01 0.03 ± 0.01 0 03 i f 0.01 Muscle 22 .27 ± 9 . 1 0 12.46 ± 4 . 6 5 9.15 ± 2.58 9.80 ± 5.11 7.87 ± 1.57 Bra in 0 58 ± 0 . 2 0 0.27 ± 0.04 0.20 i f 0.05 0.10 ± 0.03 0.10 0.03 122 Laura N . A lco rn i—11 Muscarinic Radiopharmaceutical Appendix 3. Biodis t r ibut ion P r o g r a m . Appendix 3. Biodistribution Program TEMPLATE - DOITALLwkl A L O T U S 123 template has been prepared to do the Bio distribution study. The name of the file is D O I T A L L . w k l . To use it 4 files must be prepared and stored in a subcatalog " D A T A " . The names of these files can be any combination of up to 7. characters followed by a 0 (zero ), 1, 2 and " C " . Together these characters must form a valid DOS file name. Consequently if one has D O I T A L L in directory M O D E L S and one chose the name " T E S T " the directory strings would be " B : \ M O D E L S \ D A T A \ T E S T 0 " , " B : \ M O D E L S \ D A T A \ T E S T l " etc. The model may be run by loading 123 and telling it to load file B : \ D O I T A L L (or whatever string you have for your default file source.) When 123 has loaded the model, it will automatically start and ask you for the root name so it can find these files. In the above example you would enter "TEST". The model will then proceed to load the files and prepare the output. As it is working occasionally it will change the display in location B l to notify you it is doing its job. When it has finished it will attempt to write the summary results on the file . . . \DATA\rootO where root is the name you have chosen (again, into B : \ M O D E L S \ D A T A \ T E S T O " in our example). It will then ask if you want a hard copy printout and which type of printer it must print it on. In no output is wished select N E I T H E R " and then terminate 123. The output file, rootO (Root oh ), can be loaded and edited with any word processor. 123 Laura N . A l c o r n V-^-11L Muscarinic Radiopharmaceutical Appendix 3. Biodistribution Program. File rootO - (root zero ) This is the experimental data definition file. It contains the information about the experiment, the isotope used, the number of subjects tested and the number of organs tested, etc. This information will appear in the printout or will be used to derive the working data from the recorded information found in the file 1, 2 and C . The following example will explain this input. Each line should be a separate line in the file. Only the first field of each line is used, a comma will separate optional descriptive information. The character data must be enclosed with quote (" or ' ) . The order of these items is important. The organ names can be in any order but they must contain "Blood" and "Muscle". Additionally, these two items must be spelt as shown with a Capital B and M and lowercase letters for the rest. The injection value (10) is corrected with the background value and then decayed to a value consistent with the time of first injection (7). The decay interval time is taken from the file rootC which is produced by the counter. It is assumed that the decay interval time is constant. The rest of the radioactive values will be decayed to the start time and then decayed to include these cumulative intervals. Care should be take with the injection value if a dilution factor is used. 123 will always subtract out the background before decaying the result so if a dilution factor is present the background should be added back in to this number. 124 Laura N . A l c o r n i—' 1 Muscarinic Radiopharmaceutical Appendix 3. Biodistribution Program. J. ;&-'3", . " .. Apr . 3o'iyyo " Date 1 DiscripliM-2-ibg Ligand 2 Descriptive 1-123 Nuclide 3 Descriptive 798 Half-life 4 Used in decay calculations 30 minutes time to demise 5 Descriptive 55 background 6 Used in decay calculations 0 First Decay time 7 Used in decay calculations 10 Organs 8 5 Subjects 9 330578.5 Injection 10 Used in decay calculations B l o o d 11 Live r Kidney Spleen Pancreas etc. for each organ (item 8) Stomach L u n g Heart Muscle Bra in 26 weight of each subject 11+ n 24 24 weight for each subject tested (item 9 ) 23.5 26.5 125 Laura N . A l c o r n i—11 Muscarinic Radiopharmaceutical Append ix 3. Biod is t r ibu t ion P r o g r a m . Files Rootl and Root2. These are the two weight files - the pre and post weight values. There should be exactly Subject (9) * Organ (8) items in these files. File RootC This is the file produced by the counter and edited to fit this experiment. The data consists of a series of 3 values; a count, a time interval and a reading. The first time interval is picked up and used for all the others, i.e. it assumes that all will be equal and the same as the first. If this is not the case the model could be modified to use the time intervals contained in this file. Internally the counts are ignored and only the readings are used. There should be Subject (9) * ( Organ (8) + 1 ) items in this list as there is an additional reading for the "Tail" of each subject. DONDEC93 .wkl This is the new improved Bio distribution program. The input is the same as that outlined in the previous read.me file. I have however removed the restriction on having to have a Blood and Muscle sample. If one is present the correction factor will be applied, if not it will not be applied. The correction factor is applied after all the calculations are made. 126 Laura N . A l c o r n . c=UL Muscarinic Radiopharmaceutical Appendix 3. Biodistribution Program. The program has been modified to print out the Averages, standard deviations, maximum and minimum of the Uptake/organ. I have also added the name of the files which were input to the header. To run this program in Quatro pro one must tell Qpro-v4 that you are using a 123 model with macros. To do this when Qpro is loaded go to the Tools macro menu and set Key Reader Yes. Then go to the Options Startup menu and set the Menu tree to be 123. This will turn your Qpro spread sheet into a 123 clone and you can then load and run the *.wkl models with no problems. This will not work with qpro - v5 as they have removed the 123 emulation. 127 Laura N . A l c o r n Muscarinic Radiopharmaceutical REFERENCES REFERENCES 1. Counsell R . E . , Y u T., Ranade V . V , et al. Radioiodinated bretylium analogs for myocardial scanning. J Nucl Med 1974; 15 (11):. 991-996. 2. Flanagan S.D., Storni A . A n 1251-labled binding probe for the muscarinic cholinergic receptor. Brain Res 1979; 168: 261-274. 3. 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Age-related decreases in muscarinic cholinergic receptor binding in the human brain measured with positron emission tomography (PET). J Neurosci Res 1990; 27: 569-575. 46. Narayanan N . , Derby J.-A. Effects of age on muscarinic cholinergic receptors in rat myocardium. Can J Physiol 1983; 61: 822-829. 47. PedigoN.W. Neurotransmitter receptor plasticity in aging. Life Sci 1994; 55: 1985-1991. 131 Laura N . A l c o r n Muscarinic Radiopharmaceutical R E F E R E N C E S 48. Flynn D.D. , Ferrari-DiLeo G. , Mash D . C . , Levey A.I. Differential regulation of molecular subtypes of muscarinic receptors in Alzheimer's disease. J Neurochem 1995; 64: 1888-1891. 49. Mondry A . , Bourgeois F. , Carre F. , et al. Decrease in beta(l)-adrenergic and M(2)-muscarinic receptor m R N A levels and unchanged accumulation of mRNAs coding for G(Alpha-I-2) and G(Alpha- S) proteins in rat cardiac hypertrophy. J M o l Cell Cardiol. 1995; 27: 2287-2294. 50. Fu L . - X . , Bergh C . - H . , Liang Q . - M . , et al. Diabetes-induced changes in the G i -modulated muscarinic receptor-adenylyl cyclase system in rat myocardium. Pharmacol Toxicol 1994; 75: 186-193. 51. Van Der Vliet A . , Bast A . Effect of oxidative stress on receptors and signal transmission. Chem Biol Interactions 1992; 85: 95-116. 52. Nordberg A . Effect of long-term treatment with tacrine (THA) in Alzheimer's disease as visualized by PET. Acta Neurol Scand 1993; Suppl 149: 62-65. 53. Nordberg A . Clinical studies in Alzheimer patients with positron emission tomography. Behav Brain Res 1993; 57: 215-224. 54. Delforge J., Le Guludec D. , Syrota A . , et al. Quantification of myocardial muscarinic receptors with P E T in humans. JNuc l Med 1993; 34: 981-991. 55. Wang J.-X. , Roeske W.R., Mei L . , et al. Nicotinic and muscarinic M 2 receptor alterations in the cerebral cortex of patients with senile dementia of the Alzheimer's type (SDAT). Internat Cong Ser 1987; 750: 83-86. 56. Weinberger D.R., Gibson R., Coppola R., et al. The distribution of cerebral muscarinic acetylcholine receptors in vivo in patients with dementia. A controlled study with 1 2 3 I Q N B and single photon emission computed tomography. Arch Neurol 1991; 48: 169-176. 57. Quirion R., Wilson A . , Rowe W., et al. Facilitation of acetylcholine release and cognitive performance by an M 2-muscarinic receptor antagonist in aged memory-impaired rats. JNeurosci 1995; 15: 1455-1462. 58. Hosey M . Diversity of structure, signaling and regulation within the family of muscarinic cholinergic receptors. FasebJ 1992; 6: 845-52. 59. Mertens M.J .F . , Batink H.D. , Mathy M.J . , et al. Reduced muscarinic cholinoceptor density and sensitivity in various models of experimental cardiac hypertrophy. J Aut Pharmacol. 1995; 15: 465-474. 132 Laura N . A lco rn Muscarinic Radiopharmaceutical R E F E R E N C E S 60. McGrath L . B . , Chen C , Gu J., et al. Determination of infundibular innervation and amine receptor content in cyanotic and acyanotic myocardium: relation to clinical events in Tetralogy of Fallot. Pediatr Cardiol 1991; 12: 155-160. 61. Vatner D . E . , Lee D . L . , Schwarz K.R. , et al. Impaired cardiac muscarinic receptor function in dogs with heart failure. J Clin Invest 1988; 81: 1836-1842. 62. Carre F. , Maison-Blanche P., Mansier P., et al. Phenotypic determinants of heart rate variability in cardiac hypertrophy and failure. Europ Heart J 1994; 15 (Suppl I): 58-62. 63. Hwang T . L . , Chen M . F . , Huang S.F., et al. Density of muscarinic receptors in rat myocardium during early sepsis. J Form Med Assoc. 1995; 94:655-659. 64. Valette H . , Deleuze P., Syrota A . , et al. Canine myocardial beta-adrenergic, muscarinic receptor densities after denervation: A P E T study. J N u c l M e d 1995; 36: 140-146. 65. Le Guludec D. , Delforge J., Syrota A . , et al. In vivo quantification of myocardial muscarinic receptors in heart transplant patients. Circulation 1994; 90: 172-178. 66. Fu L . - X . , Magnusson Y . , Bergh C . - H , et al. Localization of a functional autoimmune epitope on the muscarinic acetylcholine receptor-2 in patients with idiopathic dilated cardiomyopathy. J Clin Invest 1993; 91: 1964-1968. 67. Eckelman W . C . , Reba R . C . , Gibson R . E . , et al Receptor-binding radiotracers: a class of potential radiopharmaceuticals. JNuc l Med 1979; 20: 350-357. 68. Wilbur D.S., Jones D.S., Fritzberg A . R . Synthesis and radioiodinations of some aryl tin compounds for radiolabeling of monoclonal antibodies. J Label Cmpds Radiopharml9 ; XXIII (10-12): 1304-1306. 69. Winchell H.S., Baldwin R . M . , L in T H . Development of I-123-labeled amines for brain studies: Localization of 1-123 iodophenylalkyl amines in rat brain. J N u c l Med 1980; 21: 940-946. 70. Gibson R . E . , Eckelman W C , Vieras F. , Reba R C . The distribution of the muscarinic acetylcholine receptor antagonists, quinuclidinyl benzilate and quinuclidinyl benzilate methiodide (both tritiated), in rat, guinea pig, and rabbit. J N M 1979; 20: 865-870. 71. Eckelman W . C . In "Applications of Nuclear and Radiochemistry"; Lambrecht R, Marcos N . , Eds.; Pergamon Press: Elmsford, N Y , 1982: 287-298. 133 Laura N . A l c o r n Muscarinic Radiopharmaceutical R E F E R E N C E S 72. Kowalsky R.J. , Perry J.R. Radiopharmaceuticals in nuclear medicine practice. Norwalk, Appleton & Lange; 1987: 33. 73. Bylund D.B. , Yamamura H.I. Methods for receptor binding. In: Yamamura I., et al. Eds. Methods in neurotransmitter receptor analysis. New York, Raven Press Ltd.; 1990: 1-35. 74. Hrdina P.D. General principles of receptor binding. In: Boulton A . A . , Baker G.B. , Hrdina P.D., Eds. Neuromethods • 4 receptor binding. Clifton, N.J . , Humana Press. 1986: 1-22. 75. Adam M . J . , Ruth T.J. , Homma Y . , Pate B .D. Radiobromination of aromatic compounds by cleavage of aryl-tin bonds. Int J Appl Radiat Isot 1985; 36(12): 935-937. 76. Rzeszotarski W.J. , Eckelman W . C . , Francis B . E . , et al. Synthesis and evaluation of radioiodinated derivatives of l-azabicyclo[2.2.2]oct-3-yl a-hydroxy-a-(4-iodphenyl)-a-phenylacetate as potential radiopharmaceuticals. J Med Chem 1984; 27: 156-159. 77. Dougan H . , Lyster D . M . , Vincent J.S. et al. Kit type labeling of [1231] wetoiodobenzylguandine. J Label Compd Radiopharm 1988; 25: 531-544. 78. Owens J., Murray T., McCulloch J., Wyper D. Synthesis of (R,R) ' 2 3 1-QNB, a S P E C T imaging agent for cerebral muscarinic acetylcholine receptors in vivo. J Label Comp Radiopharm 1992; X X X I : 45-60. 79. Personal communication, Dr. H . Dougan 80. McRee R . C . , Boulay S.F., Sood V . K . , et al. Autoradiographic evidence that Q N B displays in vivo selectivity for the m2 subtype. Neuroimage 1995; 2: 55-62. 81. Weinberger D.R., Jones D. W., Sunderland T. et al. In vivo imaging of cerebral muscarinic receptors with 1-123 Q N B and S P E C T : Studies in normal subjects and patients with dementia. Clin Neuropharm 1992; 15(Supp 1, Pt.A): 194A-195A. 82. Gibson R . E . Quantitative changes in receptor concentration as a function of disease. In Receptor Binding Radiotracers. Vol . II. Eckelman W . C . , ed. Boca Raton, Florida. C R C Press Inc. 185-212,1982. 83. Gibson R . E . , Weckstein D.J., Jagoda E . M . , et al. The characteristics of 1-125 4-IQNB and H-3 Q N B in vivo and in vitro. JNuc l Med 1984; 25: 214-222. 134 Laura N . A lco rn Muscarinic Radiopharmaceutical R E F E R E N C E S 84. Eckelman W . C . , Eng R., Rzeszortarski W.J., Use of 3-quinuclidinyl 4-iodobenzilate as a receptor binding radiotracer. JNuc l Med 1985; 26: 637-642. 85. Jiang V . , Gibson R . E . , Rzeszortarski W.J., et al. Radioiodinated derivatives of beta adrenoceptor blockers for myocardial imaging. J Nucl Med 1978; 19: 918-924. 86. 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Binding kinetics of quinuclidinyl benzilate and methylquinuclidinyl benzilate enantiomers at neuronal (M,), cardiac (M 2 ) , and pancreatic (M 3 ) muscarinic receptors. M o l Pharmacol 1991; 40: 413-420. 138 Laura N . A l c o r n Muscarinic Radiopharmaceutical R E F E R E N C E S 119. Bonnat M . , Hercouet A . , Le Corre M . Effect of the temperature on the stoichiometry of borane dimethyl sulfide reduction of secondary and tertiary amides. Synth Comm 1991; 21: 1579-1582. 120. Lane C F . in: Brewster J .H. , Ed. Aspects of mechanism and organometallic chemistry. New York:Plenum Press, 1978: 181-198. 121. Kung H . F , Guo Y . - Z , Billings X . , et al. Preparation and biodistribution of [ 1 2 5 I]IBZM: A potential C N S D-2 dopamine receptor imaging agent. Nucl Med Biol 1988; 15: 195-201. 122. Baldwin R . M . , Zea-Ponce Y . , Al-Tikriti M . , et al. Regional brain uptake and pharmacokinetics of [123I]N-co-fluoroalkyl -2(3-carboxy-3(3-(4-iodophenyl)nortropane esters in baboons. 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